Mutant pores

ABSTRACT

The invention relates to mutant forms of Msp. The invention also relates to polynucleotide characterisation using Msp.

FIELD OF THE INVENTION

The invention relates to mutant forms of Msp. The invention also relatesto polynucleotide characterisation using Msp.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNAor RNA) sequencing and identification technologies across a wide rangeof applications. Existing technologies are slow and expensive mainlybecause they rely on amplification techniques to produce large volumesof polynucleotide and require a high quantity of specialist fluorescentchemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology.

When a potential is applied across a nanopore, there is a change in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of thenucleotide gives a current change of known signature and duration. Inthe strand sequencing method, a single polynucleotide strand is passedthrough the pore and the identities of the nucleotides are derived.Strand sequencing can involve the use of a polynucleotide bindingprotein to control the movement of the polynucleotide through the pore.

The different forms of Msp are porins from Mycobacterium smegmatis. MspAis a 157 kDa octameric porin from Mycobacterium smegmatis. Wild-typeMspA does not interact with DNA in a manner that allows the DNA to becharacterised or sequenced. The structure of MspA and the modificationsrequired for it to interact with and characterise DNA have been welldocumented (Butler, 2007, Nanopore Analysis of Nucleic Acids, Doctor ofPhilosophy Dissertation, University of Washington; Gundlach, Proc NatlAcad Sci USA. 2010 Sep. 14; 107(37):16060-5. Epub 2010 Aug. 26; andInternational Application No. PCT/GB2012/050301 (published asWO/2012/107778). Some key residues have been identified and modified toprovide observable interactions between DNA and MspA, which areessential for DNA characterisation or sequencing. In particular, Butlersupra teaches that removal of negative charge from all three ofpositions 90, 91 and 93 is required to provide observable interactionsbetween DNA and MspA.

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that novel mutants of MspAwhich retain a negative charge at position 93 may be used tocharacterise polynucleotides, such as DNA, as long as they have adecreased net negative charge in the inner lining of the cap formingregion and/or the barrel forming region. The negative charge at position93 does not affect the ability of the mutants to capturepolynucleotides, interact with polynucleotides and discriminate betweenthe nucleotides in polynucleotides.

Accordingly, the invention provides a mutant Msp monomer comprising avariant of the sequence shown in SEQ ID NO: 2, wherein the variant:

(a) does not comprise aspartic acid (D) at position 90;

(b) does not comprise aspartic acid (D) at position 91;

(c) comprises aspartic acid (D) or glutamic acid (E) at position 93; and

(d) comprises one or more modifications which decrease the net negativecharge of the inward facing amino acids in the cap forming region and/orthe barrel forming region of the monomer.

The invention also provides:

-   -   A construct comprising two or more covalently attached MspA        monomers, wherein at least one of the monomers is a mutant        monomer of the invention.    -   A polynucleotide which encodes a mutant monomer of the invention        or a construct of the invention.    -   A homo-oligomeric pore derived from Msp comprising identical        mutant monomers of the invention or identical constructs of the        invention.    -   A hetero-oligomeric pore derived from Msp comprising at least        one mutant monomer of the invention or at least one construct of        the invention.    -   A method of characterising a target polynucleotide, comprising:    -   a) contacting the polynucleotide with a pore of the invention        such that the polynucleotide moves through the pore; and    -   b) taking one or more measurements as the polynucleotide moves        with respect to the pore, wherein the measurements are        indicative of one or more characteristics of the polynucleotide,        and thereby characterising the target polynucleotide.    -   A kit for characterising a target polynucleotide comprising (a)        a pore of the invention and (b) the components of a membrane.    -   An apparatus for characterising target polynucleotides in a        sample, comprising (a) a plurality of pores of the invention        and (b) a plurality of membranes.    -   A method of characterising a target polynucleotide, comprising:    -   a) contacting the polynucleotide with a pore of the invention, a        polymerase and labelled nucleotides such that phosphate labelled        species are sequentially released when nucleotides are added to        the first polynucleotide analyte by the polymerase, wherein the        phosphate species contain a label specific for each nucleotide;        and    -   b) detecting the phosphate labelled species using the pore and        thereby characterising the polynucleotide.

DESCRIPTION OF THE FIGURES

FIG. 1 shows DNA construct X which was used in Example 1. Section a ofDNA construct X corresponds to SEQ ID NO: 26. Section b corresponds tofour iSpC3 spacers. C corresponds to the helicase enzyme T4Dda—E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutationsE94C/C109A/C136A/A360C) which can bind to the section labelled a.Section d corresponds to SEQ ID NO: 27. Section e corresponds to four5′-nitroindoles. Section f corresponds to SEQ ID NO: 28. Section gcorresponds to SEQ ID NO: 29 which is attached at its 3′ end to sixiSp18 spacers which are attached at the opposite end to two thymines and3′ cholesterol TEG (labelled h).

FIG. 2 shows DNA construct Y which was used in Example 1. Section i ofDNA construct X corresponds to 25 iSpC3 spacers, which are attached tothe 5′ end of SEQ ID NO: 3 (labelled j). Section j is the region ofconstruct Y to which the helicase enzyme T4 Dda—E94C/C109A/C136A/A360Ccan bind (labelled c). Section k corresponds to four iSp18 spacers.Section d corresponds to SEQ ID NO: 27. Section e corresponds to four5′-nitroindoles. Section 1 corresponds to SEQ ID NO: 4. Section gcorresponds to SEQ ID NO: 29 which is attached at its 3′ end to sixiSp18 spacers which are attached at the opposite end to two thymines and3′ cholesterol TEG (labelled h).

FIG. 3 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8. Sections B andC show zoomed in regions of current trace A.

FIG. 4 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct Y through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/A96D/D118R/Q126R/D134R/E139K)8. SectionsB shows a zoomed in region of current trace A.

FIG. 5 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/N102G/D118R/Q126R/D134R/E139K)8. SectionsB and C show zoomed in regions of current trace A.

FIG. 6 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/S103A/D118R/Q126R/D134R/E139K)8. SectionsB and C show zoomed in regions of current trace A.

FIG. 7 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/N108S/D118R/Q126R/D134R/E139K)8. SectionsB and C show zoomed in regions of current trace A.

FIG. 8 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/N108P/D118R/Q126R/D134R/E139K)8. SectionsB and C show zoomed in regions of current trace A.

FIG. 9 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/A96D/N108P/D118R/Q126R/D134R/E139K)8.Sections B and C show zoomed in regions of current trace A.

FIG. 10 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/A96D/N108A/D118R/Q126R/D134R/E139K)8.Sections B and C show zoomed in regions of current trace A.

FIG. 11 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanopore MspA—(G75S/G77S/L88N/I89F/D90N/D91N/D118R/Q126R/D134R/E139K)8. Sections B shows azoomed in region of current trace A.

FIG. 12 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/D90N/D91N/D118R/Q126R/D134R/E139K)8. Sections B and Cshow zoomed in regions of current trace A.

FIG. 13 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88K/D90N/D91N/I105E/D118R/Q126R/D134R/E139K)8. SectionsB shows a zoomed in region of current trace A.

FIG. 14 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/D118G/Q126R/D134R/E139K)8. Sections B andC show zoomed in regions of current trace A.

FIG. 15 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/D118N/Q126R/D134R/E139K)8. Sections B andC show zoomed in regions of current trace A.

FIG. 16 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/D118R/D134R/E139K)8. Sections B and Cshow zoomed in regions of current trace A.

FIG. 17 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88K/D90N/D91N/N108E/D118R/Q126R/D134R/E139K)8. SectionsB and C show zoomed in regions of current trace A.

FIG. 18 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct Y through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/T95E/P98K/D118R/Q126R/D134R/E139K)8.Sections B and C show zoomed in regions of current trace A.

FIG. 19 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for all three traces) of when a helicase (T4Dda—E94C/C109A/C136A/A360C) controls the translocation of the DNAconstruct X through the MspA nanoporeMspA—(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8. SectionsB and C show zoomed in regions of current trace A.

FIG. 20 shows a cartoon representation of the wild-type MspA nanopore.Region 1 corresponds to the cap forming region and includes residues1-72 and 122-184. Region 2 corresponds to the barrel forming region andincludes residues 73-82 and 112-121. Region 3 corresponds to theconstriction and loops region and includes residues 83-111.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe wild-type MspA monomer. This mutant lacks the signal sequence.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of thewild-type MspA monomer. This mutant lacks the signal sequence.

SEQ ID NOs: 3 and 4 shows polynucleotide sequences used in Example 1.

SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the polynucleotide sequence encoding the Phi29 DNApolymerase.

SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 10 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 11 shows the amino acid sequence of exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 12 shows the codon optimised polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 13 shows the amino acid sequence of the exonuclease IIIenzyme from E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides.

SEQ ID NO: 14 shows the codon optimised polynucleotide sequence derivedfrom the recJ gene from T. thermophilus. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 15 shows the amino acid sequence of the RecJ enzyme from Tthermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 16 shows the codon optimised polynucleotide sequence derivedfrom the bacteriophage lambda exo (redX) gene. It encodes thebacteriophage lambda exonuclease.

SEQ ID NO: 17 shows the amino acid sequence of the bacteriophage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 5′-3′ direction(http://www.neb.com/nebecomm/products/productM0262.asp). Enzymeinitiation on a strand preferentially requires a 5′ overhang ofapproximately 4 nucleotides with a 5′ phosphate.

SEQ ID NO: 18 shows the amino acid sequence of Hel308 Mbu.

SEQ ID NO: 19 shows the amino acid sequence of Hel308 Csy.

SEQ ID NO: 20 shows the amino acid sequence of Hel308 Tga.

SEQ ID NO: 21 shows the amino acid sequence of Hel308 Mhu.

SEQ ID NO: 22 shows the amino acid sequence of TraI Eco.

SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 26-29 shows polynucleotide sequences used in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes two or more polynucleotides, reference to “ahelicase” includes two or more helicases, reference to “a monomer”refers to two or more monomers, reference to “a pore” includes two ormore pores and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Mutant Msp Monomers

The present invention provides mutant Msp monomers. The mutant Mspmonomers may be used to form the pores of the invention. A mutant Mspmonomer is a monomer whose sequence varies from that of a wild-type Mspmonomer and which retains the ability to form a pore. Methods forconfirming the ability of mutant monomers to form pores are well-knownin the art and are discussed in more detail below.

The mutant monomers have improved polynucleotide reading properties i.e.display improved polynucleotide capture and nucleotide discrimination.In particular, pores constructed from the mutant monomers capturenucleotides and polynucleotides more easily than the wild type. Inaddition, pores constructed from the mutant monomers display anincreased current range, which makes it easier to discriminate betweendifferent nucleotides, and a reduced variance of states, which increasesthe signal-to-noise ratio. In addition, the number of nucleotidescontributing to the current as the polynucleotide moves through poresconstructed from the mutants is decreased. This makes it easier toidentify a direct relationship between the observed current as thepolynucleotide moves through the pore and the polynucleotide sequence.

A mutant monomer of the invention comprises a variant of the sequenceshown in SEQ ID NO: 2. SEQ ID NO: 2 is the wild-type MspA monomer. Avariant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequencewhich varies from that of SEQ ID NO: 2 and which retains its ability toform a pore. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into an amphiphilic layer along with other appropriate subunitsand its ability to oligomerise to form a pore may be determined. Methodsare known in the art for inserting subunits into membranes, such asamphiphilic layers. For example, subunits may be suspended in a purifiedform in a solution containing a triblock copolymer membrane such that itdiffuses to the membrane and is inserted by binding to the membrane andassembling into a functional state. Alternatively, subunits may bedirectly inserted into the membrane using the “pick and place” methoddescribed in M. A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127,6502-6503 and International Application No. PCT/GB2006/001057 (publishedas WO 2006/100484).

Positions 90 and 91

In wild-type MspA, amino acids 90 and 91 are both aspartic acid (D).These amino acids in each monomer form part of an inner constriction ofthe pore (FIG. 20). The variant does not comprise aspartic acid (D) atposition 90. The variant preferably does not comprise aspartic acid (D)or glutamic acid (E) at position 90. The variant preferably does nothave a negatively charged amino acid at position 90.

The variant does not comprise aspartic acid (D) at position 91. Thevariant preferably does not comprise aspartic acid (D) or glutamic acid(E) at position 91. The variant preferably does not have a negativelycharged amino acid at position 91.

The variant preferably comprises serine (S), glutamine (Q), leucine (L),methionine (M), isoleucine (I), alanine (A), valine (V), glycine (G),phenylalanine (F), tryptophan (W), tyrosine (Y), histidine (H),threonine (T), arginine (R), lysine (K), asparagine (N) or cysteine (C)at position 90 and/or position 91. Any combinations of these amino acidsat positions 90 and 91 are envisaged by the invention. The variantpreferably comprises asparagine (N) at position 90 and/or position 91.The variant more preferably comprises asparagine (N) at position 90 andposition 91. These amino acids are preferably inserted at position 90and/or 91 by substitution.

Position 93

In wild-type MspA, amino acid 93 is aspartic acid (D). This amino acidin each monomer also forms part of an inner constriction of the pore(FIG. 20).

The variant comprises aspartic acid (D) or glutamic acid (E) at position93. The variant therefore has a negative charge at position 93. Theglutamic acid (E) is preferably introduced by substitution.

Cap Forming Region

In wild-type MspA, amino acids 1 to 72 and 122 to 184 form the cap ofthe pore (FIG. 20). Of these amino acids, V9, Q12, D13, R14, T15, W40,I49, P53, G54, D56, E57, E59, T61, E63, Y66, Q67, 168, F70, P123, 1125,Q126, E127, V128, A129, T130, F131, S132, V133, D134, S136, G137, E139,V144, H148, T150, V151, T152, F163, R165, 1167, S169, T170 and S173 faceinwards into the channel of the pore.

Barrel Forming Region

In wild-type MspA, amino acids 73 to 82 and 112 to 121 form the barrelof the pore (FIG. 20). Of these amino acids, S73, G75, G77, N79, S81,G112, S114, S116, D118 and G120 face inwards into the channel of thepore. S73, G75, G77, N79, S81 face inwards in the downwards strand andG112, S114, S116, D118 and G120 face inwards in the upwards strand.

Decreased Net Negative Charge

The variant comprises one or more modifications which decrease the netnegative charge of the inward facing amino acids in the cap formingregion and/or the barrel forming region of the monomer. The variantpreferably comprises two or more modifications which decrease the netnegative charge of the inward facing amino acids in the cap formingregion and the barrel forming region of the monomer.

The variant may comprise any number of modifications, such as 1 or more,2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, or 40or more modifications.

The net negative charge may be decreased by any means known in the art.The net negative charge is decreased in a manner that does not interferewith the ability of the mutant monomer to form a pore. This can bemeasured as discussed above.

The net negative charge of the inward facing amino acids in the capforming region and/or the barrel forming region is decreased. This meansthat the inward facing amino acids in the cap forming region and/or thebarrel forming region comprise fewer negatively charged amino acids thanin SEQ ID NO: 2 and/or comprises more positively charged amino acidsthan in SEQ ID NO: 2. The one or more modifications may lead to a netpositive charge in the inward facing amino acids in the cap formingregion and/or the barrel forming region

The net charge can be measured using methods known in the art. Forinstance, the isolectric point may be used to define the net charge ofthe inward facing amino acids in the cap forming region and/or thebarrel forming region.

The one or more modifications are preferably one or more deletions ofnegatively charged amino acids. Removal of one or more negativelycharged amino acids reduces the net negative charge of the inward facingamino acids in the cap forming region and/or barrel forming region. Anegatively charged amino acid is an amino acid with a net negativecharge. Negatively charged amino acids include, but are not limited to,aspartic acid (D) and glutamic acid (E). Methods for deleting aminoacids from proteins, such as MspA monomers, are well known in the art.

The one or more modifications are preferably one or more substitutionsof negatively charged amino acids with one or more positively charged,uncharged, non-polar and/or aromatic amino acids. A positively chargedamino acid is an amino acid with a net positive charge. The positivelycharged amino acid(s) can be naturally-occurring ornon-naturally-occurring. The positively charged amino acid(s) may besynthetic or modified. For instance, modified amino acids with a netpositive charge may be specifically designed for use in the invention. Anumber of different types of modification to amino acids are well knownin the art.

Preferred naturally-occurring positively charged amino acids include,but are not limited to, histidine (H), lysine (K) and arginine (R). Anynumber and combination of H, K and/or R may be substituted for theinward facing amino acids in the cap forming region and/or barrelforming region.

The uncharged amino acids, non-polar amino acids and/or aromatic aminoacids can be naturally occurring or non-naturally-occurring. They may besynthetic or modified. Uncharged amino acids have no net charge.Suitable uncharged amino acids include, but are not limited to, cysteine(C), serine (S), threonine (T), methionine (M), asparagines (N) andglutamine (Q). Non-polar amino acids have non-polar side chains.Suitable non-polar amino acids include, but are not limited to, glycine(G), alanine (A), proline (P), isoleucine (I), leucine (L) and valine(V). Aromatic amino acids have an aromatic side chain. Suitable aromaticamino acids include, but are not limited to, histidine (H),phenylalanine (F), tryptophan (W) and tyrosine (Y). Any number andcombination of these amino acids may be substituted into the inwardfacing amino acids in the cap forming region and/or the barrel formingregion.

The one or more negatively charged amino acids are preferablysubstituted with alanine (A), valine (V), asparagine (N) or glycine (G).Preferred substitutions include, but are not limited to, substitution ofD with A, substitution of D with V, substitution of D with N andsubstitution of D with G.

The one or more modifications are preferably one or more introductionsof positively charged amino acids. The introduction of positive chargedecreases the net negative charge. The one or more positively chargedamino acids may be introduced by addition or substitution. Any aminoacid may be substituted with a positively charged amino acid. One ormore uncharged amino acids, non-polar amino acids and/or aromatic aminoacids may be substituted with one or more positively charged aminoacids. Any number of positively charged amino acids may be introduced.

Wild-type MspA comprises a polar glutamine (Q) at position 126. The oneor more modifications preferably reduce the net negative charge atposition 126. The one or more modifications preferably increase the netpositive charge at positions 126. This can be achieved by replacing thepolar amino acid at position 126 or an adjacent or a nearby inwardfacing amino acid with a positively charged amino acid. The variantpreferably comprises a positively charged amino acid at position 126.The variant preferably comprises a positively charged amino acid at oneor more of positions 123, 125, 127 and 128. The variant may comprise anynumber and combination of positively charged amino acids at positions123, 125, 127 and 128. The positively charged amino acid(s) may beintroduced by addition or substitution.

The one or more modifications are preferably one or more introductionsof positively charged amino acids which neutralise one or morenegatively charged amino acids. The neutralisation of negative chargedecreases the net negative charge. The one or more positively chargedamino acids may be introduced by addition or substitution. Any aminoacid may be substituted with a positively charged amino acid. One ormore uncharged amino acids, non-polar amino acids and/or aromatic aminoacids may be substituted with one or more positively charged aminoacids. Any number of positively charged amino acids may be introduced.The number is typically the same as the number of negatively chargedamino acids in the inward facing amino acids in the cap forming regionand/or the barrel forming region.

The one or more positively charged amino acids may be introduced at anyposition in the cap forming region and/or the barrel forming region aslong as they neutralise the negative charge of the one or morenegatively charged amino acids. To effectively neutralise the negativecharge, there is typically 5 or fewer amino acids in the variant betweeneach positively charged amino acid that is introduced and the negativelycharged amino acid it is neutralising. There is preferably 4 or fewer, 3or fewer or 2 or fewer amino acids in the variant between eachpositively charged amino acid that is introduced and the negativelycharged amino acid it is neutralising. There is more preferably twoamino acids in the variant between each positively charged amino acidthat is introduced and the negatively charged amino acid it isneutralising. Each positively charged amino acid is most preferablyintroduced adjacent in the variant to the negatively charged amino acidit is neutralising.

The one or more positively charged amino acids may be introduced at anyposition in the inward facing amino acids in the cap forming regionand/or the barrel forming region as long as they neutralise the negativecharge of the one or more negatively charged amino acids. To effectivelyneutralise the negative charge, there is typically 5 or fewer inwardfacing amino acids between each positively charged amino acid that isintroduced and the negatively charged amino acid it is neutralising.There is preferably 4 or fewer, 3 or fewer or 2 or fewer inward facingamino acids between each positively charged amino acid that isintroduced and the negatively charged amino acid it is neutralising.There is more preferably one inward facing amino acid between eachpositively charged amino acid that is introduced and the negativelycharged amino acid it is neutralising. Each positively charged aminoacid is most preferably introduced at the inward facing positionadjacent to the negatively charged amino acid it is neutralising.

Wild-type MspA comprises aspartic acid (D) at positions 118 and 134 andglutamic acid (E) at position 139. Amino acid 118 in each monomer ispresent within the barrel of the pore (FIG. 20). The variant preferablycomprises a positively charged amino acid at one or more of positions114, 116, 120, 123, 70, 73, 75, 77 and 79. Positive charges at one ormore of these positions neutralise the negative charge at position 118.Positively charged amino acids may present at any number and combinationof positions 114, 116, 120, 123, 70, 73, 75, 77 and 79. The amino acidsmay be introduced by addition or substitution.

Amino acids 134 and 139 in each monomer are part of the cap (FIG. 20).The variant comprises a positively charged amino acid at one or more ofpositions 129, 132, 136, 137, 59, 61 and 63. Positive charges at one ormore of these positions neutralise the negative charge at position 134.Positively charged amino acids may present at any number and combinationof positions 129, 132, 136, 137, 59, 61 and 63. The amino acids may beintroduced by addition or substitution.

The variant preferably comprises a positively charged amino acid at oneor more of positions 137, 138, 141, 143, 45, 47, 49 and 51. Positivecharges at one or more of these positions neutralise the negative chargeat position 139. Positively charged amino acids may present at anynumber and combination of positions 137, 138, 141, 143, 45, 47, 49 and51. The amino acids may be introduced by addition or substitution.

Positions 118, 126, 134 and 139

The one or more modifications preferably reduce the net negative chargeat one or more of positions 118, 126, 134 and 139. The one or moremodifications preferably reduce the net negative charge at 118; 126;134; 139; 118 and 126; 118 and 134; 118 and 139; 126 and 134; 126 and139; 134 and 139; 118, 126 and 134; 118, 126 and 139; 118, 134 and 139;126, 134 and 139; or 118, 126, 134 and 139.

The variant preferably does not comprise aspartic acid (D) or glutamicacid (E) at one or more of positions 118, 126, 134 and 139. The variantpreferably does not comprise aspartic acid (D) or glutamic acid (E) atany of the combination of positions 118, 126, 134 and 139 disclosedabove. The variant more preferably comprises arginine (R), glycine (G)or asparagine (N) at one or more of positions 118, 126, 134 and 139,such as any of the combinations of positions 118, 126, 134 and 139disclosed above. The variant most preferably comprises D118R, Q126R,D134R and E139K.

Methods for introducing or substituting naturally-occurring amino acidsare well known in the art. For instance, methionine (M) may besubstituted with arginine (R) by replacing the codon for methionine(ATG) with a codon for arginine (CGT) at the relevant position in apolynucleotide encoding the mutant monomer. The polynucleotide can thenbe expressed as discussed below.

Methods for introducing or substituting non-naturally-occurring aminoacids are also well known in the art. For instance,non-naturally-occurring amino acids may be introduced by includingsynthetic aminoacyl-tRNAs in the IVTT system used to express the mutantmonomer. Alternatively, they may be introduced by expressing the mutantmonomer in E. coli that are auxotrophic for specific amino acids in thepresence of synthetic (i.e. non-naturally-occurring) analogues of thosespecific amino acids. They may also be produced by naked ligation if themutant monomer is produced using partial peptide synthesis.

The one or more modifications are preferably one or more chemicalmodifications of one or more negatively charged amino acids whichneutralise their negative charge. For instance, the one or morenegatively charged amino acids may be reacted with a carbodiimide.

Other Modifications

The variant preferably comprises one or more of:

(a) serine (S) at position 75;

(b) serine (S) at position 77; and

(c) asparagine (N) or lysine (K) at position 88.

The variant may comprise any number and combination of (a) to (c),including (a), (b), (c), (a) and (b), (b) and (c), (a) and (c) and (a),(b) and (c). The variant preferably comprises G75S, G77S and L88N.

The variant most preferably comprises G75S, G77S, L88N, D90N, D91N,D118R, Q126R, D134R and E139K.

The variant preferably further comprises one or more of:

(d) phenylalanine (F) at position 89;

(e) glutamic acid (E) at position 95 and lysine (K) at position 98;

(f) aspartic acid (D) at position 96;

(g) glycine (G) at position 102;

(h) alanine (A) at position 103; and

(i) alanine (A), serine (S) or proline (P) at position 108.

The may comprise any number and combination of (d) to (i).

Variants

In addition to the specific mutations discussed above, the variant mayinclude other mutations. Over the entire length of the amino acidsequence of SEQ ID NO: 2, a variant will preferably be at least 50%homologous to that sequence based on amino acid identity. Morepreferably, the variant may be at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90% andmore preferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 2 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 100 or more, for example 125,150, 175 or 200 or more, contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et at (1984) Nucleic Acids Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet at (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the mature form of the wild-type MspA monomer. Thevariant may comprise any of the mutations in the MspB, C or D monomerscompared with MspA. The mature forms of MspB, C and D are shown in SEQID NOs: 5 to 7. In particular, the variant may comprise the followingsubstitution present in MspB: A138P. The variant may comprise one ormore of the following substitutions present in MspC: A96G, N102E andA138P. The variant may comprise one or more of the following mutationspresent in MspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T,I49H, 168V, D91G, A96Q, N102D, S103T, V1041, S136K and G141A. Thevariant may comprise combinations of one or more of the mutations andsubstitutions from Msp B, C and D.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 1below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 1.

TABLE 1 Chemical properties of amino acids Ala aliphatic, hydrophobic,Met hydrophobic, neutral neutral Cys polar, hydrophobic, Asn polar,hydrophilic, neutral neutral Asp polar, hydrophilic, Pro hydrophobic,neutral charged (−) Glu polar, hydrophilic, Gln polar, hydrophilic,charged (−) neutral Phe aromatic, hydrophobic, Arg polar, hydrophilic,neutral charged (+) Gly aliphatic, neutral Ser polar, hydrophilic,neutral His aromatic, polar, Thr polar, hydrophilic, hydrophilic,charged (+) neutral Ile aliphatic, hydrophobic, Val aliphatic,hydrophobic, neutral neutral Lys polar, hydrophilic, Trp aromatic,hydrophobic, charged(+) neutral Leu aliphatic, hydrophobic, Tyraromatic, polar, neutral hydrophobic

TABLE 2 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. Fragments may be at least 50, 100, 150 or 200amino acids in length. Such fragments may be used to produce the pores.A fragment preferably comprises the pore forming domain of SEQ ID NO: 2.Fragments must include one of residues 88, 90, 91, 105, 118 and 134 ofSEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91,105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 2 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 2 that are responsible for pore formation. The pore formingability of Msp, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 2 typically comprises the regionsin SEQ ID NO: 2 that form β-sheets. One or more modifications can bemade to the regions of SEQ ID NO: 2 that form β-sheets as long as theresulting variant retains its ability to form a pore. A variant of SEQID NO: 2 preferably includes one or more modifications, such assubstitutions, additions or deletions, within its α-helices and/or loopregions.

The monomers derived from Msp may be modified to assist theiridentification or purification, for example by the addition of astreptavidin tag or by the addition of a signal sequence to promotetheir secretion from a cell where the monomer does not naturally containsuch a sequence. Other suitable tags are discussed in more detail below.The monomer may be labelled with a revealing label. The revealing labelmay be any suitable label which allows the monomer to be detected.Suitable labels are described below.

The monomer derived from Msp may also be produced using D-amino acids.For instance, the monomer derived from Msp may comprise a mixture ofL-amino acids and D-amino acids. This is conventional in the art forproducing such proteins or peptides.

The monomer derived from Msp contains one or more specific modificationsto facilitate nucleotide discrimination. The monomer derived from Mspmay also contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe monomer derived from Msp. Such modifications include, for example,reductive alkylation of amino acids by reaction with an aldehydefollowed by reduction with NaBH₄, amidination with methylacetimidate oracylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methodsknown in the art. The monomer derived from Msp may be made syntheticallyor by recombinant means. For example, the monomer may be synthesized byin vitro translation and transcription (IVTT). Suitable methods forproducing pores and monomers are discussed in International ApplicationNos. PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679(published as WO 2010/004265) or PCT/GB10/000133 (published as WO2010/086603).

In some embodiments, the mutant monomer is chemically modified. Themutant monomer can be chemically modified in any way and at any site.The mutant monomer is preferably chemically modified by attachment of amolecule to one or more cysteines (cysteine linkage), attachment of amolecule to one or more lysines, attachment of a molecule to one or morenon-natural amino acids, enzyme modification of an epitope ormodification of a terminus. Suitable methods for carrying out suchmodifications are well-known in the art. The mutant monomer may bechemically modified by the attachment of any molecule. For instance, themutant monomer may be chemically modified by attachment of a dye or afluorophore.

In some embodiments, the mutant monomer is chemically modified with amolecular adaptor that facilitates the interaction between a porecomprising the monomer and a target nucleotide or target polynucleotidesequence. The presence of the adaptor improves the host-guest chemistryof the pore and the nucleotide or polynucleotide sequence and therebyimproves the sequencing ability of pores formed from the mutant monomer.The principles of host-guest chemistry are well-known in the art. Theadaptor has an effect on the physical or chemical properties of the porethat improves its interaction with the nucleotide or polynucleotidesequence. The adaptor may alter the charge of the barrel or channel ofthe pore or specifically interact with or bind to the nucleotide orpolynucleotide sequence thereby facilitating its interaction with thepore.

The molecular adaptor is preferably a cyclic molecule, a cyclodextrin, aspecies that is capable of hybridization, a DNA binder or interchelator,a peptide or peptide analogue, a synthetic polymer, an aromatic planarmolecule, a small positively-charged molecule or a small moleculecapable of hydrogen-bonding.

The adaptor may be cyclic. A cyclic adaptor preferably has the samesymmetry as the pore. The adaptor preferably has eight-fold symmetrysince Msp typically has eight subunits around a central axis. This isdiscussed in more detail below.

The adaptor typically interacts with the nucleotide or polynucleotidesequence via host-guest chemistry. The adaptor is typically capable ofinteracting with the nucleotide or polynucleotide sequence. The adaptorcomprises one or more chemical groups that are capable of interactingwith the nucleotide or polynucleotide sequence. The one or more chemicalgroups preferably interact with the nucleotide or polynucleotidesequence by non-covalent interactions, such as hydrophobic interactions,hydrogen bonding, Van der Waal's forces, π-cation interactions and/orelectrostatic forces. The one or more chemical groups that are capableof interacting with the nucleotide or polynucleotide sequence arepreferably positively charged. The one or more chemical groups that arecapable of interacting with the nucleotide or polynucleotide sequencemore preferably comprise amino groups. The amino groups can be attachedto primary, secondary or tertiary carbon atoms. The adaptor even morepreferably comprises a ring of amino groups, such as a ring of 6, 7 or 8amino groups. The adaptor most preferably comprises a ring of eightamino groups. A ring of protonated amino groups may interact withnegatively charged phosphate groups in the nucleotide or polynucleotidesequence.

The correct positioning of the adaptor within the pore can befacilitated by host-guest chemistry between the adaptor and the porecomprising the mutant monomer. The adaptor preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore. The adaptor more preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore via non-covalent interactions, such ashydrophobic interactions, hydrogen bonding, Van der Waal's forces,π-cation interactions and/or electrostatic forces. The chemical groupsthat are capable of interacting with one or more amino acids in the poreare typically hydroxyls or amines. The hydroxyl groups can be attachedto primary, secondary or tertiary carbon atoms. The hydroxyl groups mayform hydrogen bonds with uncharged amino acids in the pore. Any adaptorthat facilitates the interaction between the pore and the nucleotide orpolynucleotide sequence can be used.

Suitable adaptors include, but are not limited to, cyclodextrins, cyclicpeptides and cucurbiturils. The adaptor is preferably a cyclodextrin ora derivative thereof. The cyclodextrin or derivative thereof may be anyof those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am.Chem. Soc. 116, 6081-6088. The adaptor is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). The guanidinogroup in gu₇-βCD has a much higher pKa than the primary amines inam₇-βCD and so it more positively charged. This gu₇-βCD adaptor may beused to increase the dwell time of the nucleotide in the pore, toincrease the accuracy of the residual current measured, as well as toincrease the base detection rate at high temperatures or low dataacquisition rates.

If a succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker isused as discussed in more detail below, the adaptor is preferablyheptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropanoyl-β-cyclodextrin(am₆amPDP₁-βCD).

More suitable adaptors include γ-cyclodextrins, which comprise 8 sugarunits (and therefore have eight-fold symmetry). The γ-cyclodextrin maycontain a linker molecule or may be modified to comprise all or more ofthe modified sugar units used in the β-cyclodextrin examples discussedabove.

The molecular adaptor is preferably covalently attached to the mutantmonomer. The adaptor can be covalently attached to the pore using anymethod known in the art. The adaptor is typically attached via chemicallinkage. If the molecular adaptor is attached via cysteine linkage, theone or more cysteines have preferably been introduced to the mutant bysubstitution. The mutant monomers of the invention can of coursecomprise a cysteine residue at one or more of positions 88, 90, 91, 103and 105. The mutant monomer may be chemically modified by attachment ofa molecular adaptor to one or more, such as 2, 3, 4 or 5, of thesecysteines. Alternatively, the mutant monomer may be chemically modifiedby attachment of a molecule to one or more cysteines introduced at otherpositions. The molecular adaptor is preferably attached to one or moreof positions 90, 91 and 103 of SEQ ID NO: 2.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the mutant monomer before a linker is attached. The moleculemay be attached directly to the mutant monomer. The molecule ispreferably attached to the mutant monomer using a linker, such as achemical crosslinker or a peptide linker.

Suitable chemical crosslinkers are well-known in the art. Preferredcrosslinkers include 2,5-dioxopyrrolidin-1-yl3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-1-yl8-(pyridin-2-yldisulfanyl)octananoate. The most preferred crosslinker issuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, themolecule is covalently attached to the bifunctional crosslinker beforethe molecule/crosslinker complex is covalently attached to the mutantmonomer but it is also possible to covalently attach the bifunctionalcrosslinker to the monomer before the bifunctional crosslinker/monomercomplex is attached to the molecule.

The linker is preferably resistant to dithiothreitol (DTT). Suitablelinkers include, but are not limited to, iodoacetamide-based andMaleimide-based linkers.

In other embodiment, the monomer may be attached to a polynucleotidebinding protein. This forms a modular sequencing system that may be usedin the methods of sequencing of the invention. Polynucleotide bindingproteins are discussed below.

The polynucleotide binding protein is preferably covalently attached tothe mutant monomer. The protein can be covalently attached to themonomer using any method known in the art. The monomer and protein maybe chemically fused or genetically fused. The monomer and protein aregenetically fused if the whole construct is expressed from a singlepolynucleotide sequence. Genetic fusion of a monomer to a polynucleotidebinding protein is discussed in International Application No.PCT/GB09/001679 (published as WO 2010/004265).

If the polynucleotide binding protein is attached via cysteine linkage,the one or more cysteines have preferably been introduced to the mutantby substitution. The mutant monomers of the invention can of coursecomprise cysteine residues at one or more of positions 10 to 15, 51 to60, 136 to 139 and 168 to 172. These positions are present in loopregions which have low conservation amongst homologues indicating thatmutations or insertions may be tolerated. They are therefore suitablefor attaching a polynucleotide binding protein. The reactivity ofcysteine residues may be enhanced by modification as described above.

The polynucleotide binding protein may be attached directly to themutant monomer or via one or more linkers. The molecule may be attachedto the mutant monomer using the hybridization linkers described inInternational Application No. PCT/GB10/000132 (published as WO2010/086602). Alternatively, peptide linkers may be used. Peptidelinkers are amino acid sequences. The length, flexibility andhydrophilicity of the peptide linker are typically designed such that itdoes not to disturb the functions of the monomer and molecule. Preferredflexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10or 16, serine and/or glycine amino acids. More preferred flexiblelinkers include (SG)₁, (SG)₂, (SG)₃, (SG)₄, (SG)₅ and (SG)₈ wherein S isserine and G is glycine. Preferred rigid linkers are stretches of 2 to30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigidlinkers include (P)₁₂ wherein P is proline.

The mutant monomer may be chemically modified with a molecular adaptorand a polynucleotide binding protein.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the monomer before a linker is attached.

The molecule (with which the monomer is chemically modified) may beattached directly to the monomer or attached via a linker as disclosedin International Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603).

Any of the proteins described herein, such as the mutant monomers andpores of the invention, may be modified to assist their identificationor purification, for example by the addition of histidine residues (ahis tag), aspartic acid residues (an asp tag), a streptavidin tag, aflag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of asignal sequence to promote their secretion from a cell where thepolypeptide does not naturally contain such a sequence. An alternativeto introducing a genetic tag is to chemically react a tag onto a nativeor engineered position on the protein. An example of this would be toreact a gel-shift reagent to a cysteine engineered on the outside of theprotein. This has been demonstrated as a method for separating hemolysinhetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

Any of the proteins described herein, such as the mutant monomers andpores of the invention, may be labelled with a revealing label. Therevealing label may be any suitable label which allows the protein to bedetected. Suitable labels include, but are not limited to, fluorescentmolecules, radioisotopes e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

Any of the proteins described herein, such as the monomers or pores ofthe invention, may be made synthetically or by recombinant means. Forexample, the protein may be synthesized by in vitro translation andtranscription (IVTT). The amino acid sequence of the protein may bemodified to include non-naturally occurring amino acids or to increasethe stability of the protein. When a protein is produced by syntheticmeans, such amino acids may be introduced during production. The proteinmay also be altered following either synthetic or recombinantproduction.

Proteins may also be produced using D-amino acids. For instance, theprotein may comprise a mixture of L-amino acids and D-amino acids. Thisis conventional in the art for producing such proteins or peptides.

The protein may also contain other non-specific modifications as long asthey do not interfere with the function of the protein. A number ofnon-specific side chain modifications are known in the art and may bemade to the side chains of the protein(s). Such modifications include,for example, reductive alkylation of amino acids by reaction with analdehyde followed by reduction with NaBH₄, amidination withmethylacetimidate or acylation with acetic anhydride.

Any of the proteins described herein, including the monomers and poresof the invention, can be produced using standard methods known in theart. Polynucleotide sequences encoding a protein may be derived andreplicated using standard methods in the art. Polynucleotide sequencesencoding a protein may be expressed in a bacterial host cell usingstandard techniques in the art. The protein may be produced in a cell byin situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide. These methods aredescribed in Sambrook, J. and Russell, D. (2001). Molecular Cloning: ALaboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

Proteins may be produced in large scale following purification by anyprotein liquid chromatography system from protein producing organisms orafter recombinant expression. Typical protein liquid chromatographysystems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system.

Constructs

The invention also provides a construct comprising two or morecovalently attached MspA monomers, wherein at least one of the monomersis a mutant monomer of the invention. The construct of the inventionretains its ability to form a pore. This may be determined as discussedabove. One or more constructs of the invention may be used to form poresfor characterising, such as sequencing, polynucleotides. The constructmay comprise at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9 or at least 10 monomers. Theconstruct preferably comprises two monomers. The two or more monomersmay be the same or different.

The construct may comprise one or more monomers which are not mutantmonomers of the invention. MspA mutant monomers which are non mutantmonomers of the invention comprise comparative variants of SEQ ID NO: 2.At least one monomer in the construct may comprise a comparative variantof the sequence shown in SEQ ID NO: 2 which comprises D90N, D91N, D93N,D118R, D134R and E139K. At least one monomer in the construct may be anyof the monomers disclosed in International Application No.PCT/GB2012/050301 (published as WO/2012/107778), including thosecomprising a comparative variant of the sequence shown in SEQ ID NO: 2which comprises G75S, G77S, L88N, D90N, D91N, D93N, D118R, Q126R, D134Rand E139K. A comparative variant of SEQ ID NO: 2 is at least 50%homologous to SEQ ID NO: 2 over its entire sequence based on amino acididentity. More preferably, the comparative variant may be at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% and more preferably at least 95%, 97% or 99%homologous based on amino acid identity to the amino acid sequence ofSEQ ID NO: 2 over the entire sequence.

At least one monomer in the construct is a mutant monomer of theinvention. All of the monomers in the construct may be a mutant monomerof the invention. The mutant monomers may be the same or different. In amore preferred embodiment, the construct comprises two monomers of theinvention.

The monomers in the construct are preferably genetically fused. Monomersare genetically fused if the whole construct is expressed from a singlepolynucleotide sequence. The coding sequences of the monomers may becombined in any way to form a single polynucleotide sequence encodingthe construct.

The monomers may be genetically fused in any configuration. The monomersmay be fused via their terminal amino acids. For instance, the aminoterminus of the one monomer may be fused to the carboxy terminus ofanother monomer. The second and subsequent monomers in the construct (inthe amino to carboxy direction) may comprise a methionine at their aminoterminal ends (each of which is fused to the carboxy terminus of theprevious monomer). For instance, if M is a monomer (without an aminoterminal methionine) and mM is a monomer with an amino terminalmethionine, the construct may comprise the sequence M-mM, M-mM-mM orM-mM-mM-mM. The presences of these methionines typically results fromthe expression of the start codons (i.e. ATGs) at the 5′ end of thepolynucleotides encoding the second or subsequent monomers within thepolynucleotide encoding entire construct. The first monomer in theconstruct (in the amino to carboxy direction) may also comprise amethionine (e.g. mM-mM, mM-mM-mM or mM-mM-mM-mM).

The two or more monomers may be genetically fused directly together. Themonomers are preferably genetically fused using a linker. The linker maybe designed to constrain the mobility of the monomers. Preferred linkersare amino acid sequences (i.e. peptide linkers). Any of the peptidelinkers discussed above may be used.

In another preferred embodiment, the monomers are chemically fused. Twomonomers are chemically fused if the two parts are chemically attached,for instance via a chemical crosslinker. Any of the chemicalcrosslinkers discussed above may be used. The linker may be attached toone or more cysteine residues introduced into a mutant monomer of theinvention. Alternatively, the linker may be attached to a terminus ofone of the monomers in the construct.

If a construct contains different monomers, crosslinkage of monomers tothemselves may be prevented by keeping the concentration of linker in avast excess of the monomers. Alternatively, a “lock and key” arrangementmay be used in which two linkers are used. Only one end of each linkermay react together to form a longer linker and the other ends of thelinker each react with a different monomers. Such linkers are describedin International Application No. PCT/GB10/000132 (published as WO2010/086602).

Polynucleotides

The present invention also provides polynucleotide sequences whichencode a mutant monomer of the invention. The mutant monomer may be anyof those discussed above. The polynucleotide sequence preferablycomprises a sequence at least 50%, 60%, 70%, 80%, 90% or 95% homologousbased on nucleotide identity to the sequence of SEQ ID NO: 1 over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95% nucleotide identity over a stretch of 300 or more, forexample 375, 450, 525 or 600 or more, contiguous nucleotides (“hardhomology”). Homology may be calculated as described above. Thepolynucleotide sequence may comprise a sequence that differs from SEQ IDNO: 1 on the basis of the degeneracy of the genetic code.

The present invention also provides polynucleotide sequences whichencode any of the genetically fused constructs of the invention. Thepolynucleotide preferably comprises two or more variants of the sequenceshown in SEQ ID NO: 1. The polynucleotide sequence preferably comprisestwo or more sequences having at least 50%, 60%, 70%, 80%, 90% or 95%homology to SEQ ID NO: 1 based on nucleotide identity over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95% nucleotide identity over a stretch of 600 or more, for example 750,900, 1050 or 1200 or more, contiguous nucleotides (“hard homology”).Homology may be calculated as described above.

Polynucleotide sequences may be derived and replicated using standardmethods in the art. Chromosomal DNA encoding wild-type Msp may beextracted from a pore producing organism, such as Mycobacteriumsmegmatis. The gene encoding the pore subunit may be amplified using PCRinvolving specific primers. The amplified sequence may then undergosite-directed mutagenesis. Suitable methods of site-directed mutagenesisare known in the art and include, for example, combine chain reaction.Polynucleotides encoding a construct of the invention can be made usingwell-known techniques, such as those described in Sambrook, J. andRussell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The resulting polynucleotide sequence may then be incorporated into arecombinant replicable vector such as a cloning vector. The vector maybe used to replicate the polynucleotide in a compatible host cell. Thuspolynucleotide sequences may be made by introducing a polynucleotideinto a replicable vector, introducing the vector into a compatible hostcell, and growing the host cell under conditions which bring aboutreplication of the vector. The vector may be recovered from the hostcell. Suitable host cells for cloning of polynucleotides are known inthe art and described in more detail below.

The polynucleotide sequence may be cloned into suitable expressionvector. In an expression vector, the polynucleotide sequence istypically operably linked to a control sequence which is capable ofproviding for the expression of the coding sequence by the host cell.Such expression vectors can be used to express a pore subunit.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. Multiple copies of the same or different polynucleotidesequences may be introduced into the vector.

The expression vector may then be introduced into a suitable host cell.Thus, a mutant monomer or construct of the invention can be produced byinserting a polynucleotide sequence into an expression vector,introducing the vector into a compatible bacterial host cell, andgrowing the host cell under conditions which bring about expression ofthe polynucleotide sequence. The recombinantly-expressed monomer orconstruct may self-assemble into a pore in the host cell membrane.Alternatively, the recombinant pore produced in this manner may beremoved from the host cell and inserted into another membrane. Whenproducing pores comprising at least two different monomers orconstructs, the different monomers or constructs may be expressedseparately in different host cells as described above, removed from thehost cells and assembled into a pore in a separate membrane, such as arabbit cell membrane.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide sequence and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example a tetracycline resistance gene. Promoters and otherexpression regulation signals may be selected to be compatible with thehost cell for which the expression vector is designed. A T7, trc, lac,ara or λ_(L) promoter is typically used.

The host cell typically expresses the monomer or construct at a highlevel. Host cells transformed with a polynucleotide sequence will bechosen to be compatible with the expression vector used to transform thecell. The host cell is typically bacterial and preferably Escherichiacoli. Any cell with a λ DE3 lysogen, for example C41 (DE3), BL21 (DE3),JM109 (DE3), B834 (DE3), TUNER, Origami and Origami B, can express avector comprising the T7 promoter. In addition to the conditions listedabove any of the methods cited in Proc Natl Acad Sci USA. 2008 Dec. 30;105(52):20647-52 may be used to express the Msp proteins.

The invention also comprises a method of producing a mutant monomer ofthe invention or a construct of the invention. The method comprisesexpressing a polynucleotide of the invention in a suitable host cell.The polynucleotide is preferably part of a vector and is preferablyoperably linked to a promoter.

Pores

The invention also provides various pores. The pores of the inventionare ideal for characterising, such as sequencing, polynucleotidesequences because they can discriminate between different nucleotideswith a high degree of sensitivity. The pores can surprisinglydistinguish between the four nucleotides in DNA and RNA. The pores ofthe invention can even distinguish between methylated and unmethylatednucleotides. The base resolution of pores of the invention issurprisingly high. The pores show almost complete separation of all fourDNA nucleotides. The pores further discriminate between deoxycytidinemonophosphate (dCMP) and methyl-dCMP based on the dwell time in the poreand the current flowing through the pore.

The pores of the invention can also discriminate between differentnucleotides under a range of conditions. In particular, the pores willdiscriminate between nucleotides under conditions that are favourable tothe characterising, such as sequencing, of nucleic acids. The extent towhich the pores of the invention can discriminate between differentnucleotides can be controlled by altering the applied potential, thesalt concentration, the buffer, the temperature and the presence ofadditives, such as urea, betaine and DTT. This allows the function ofthe pores to be fine-tuned, particularly when sequencing. This isdiscussed in more detail below. The pores of the invention may also beused to identify polynucleotide polymers from the interaction with oneor more monomers rather than on a nucleotide by nucleotide basis.

A pore of the invention may be isolated, substantially isolated,purified or substantially purified. A pore of the invention is isolatedor purified if it is completely free of any other components, such aslipids or other pores. A pore is substantially isolated if it is mixedwith carriers or diluents which will not interfere with its intendeduse. For instance, a pore is substantially isolated or substantiallypurified if it is present in a form that comprises less than 10%, lessthan 5%, less than 2% or less than 1% of other components, such astriblock copolymers, lipids or other pores. Alternatively, a pore of theinvention may be present in a membrane. Suitable membranes are discussedbelow.

A pore of the invention may be present as an individual or single pore.Alternatively, a pore of the invention may be present in a homologous orheterologous population of two or more pores.

Homo-Oligomeric Pores

The invention also provides a homo-oligomeric pore derived from Mspcomprising identical mutant monomers of the invention. Thehomo-oligomeric pore may comprise any of the mutants of the invention.The homo-oligomeric pore of the invention is ideal for characterising,such as sequencing, polynucleotides. The homo-oligomeric pore of theinvention may have any of the advantages discussed above.

The homo-oligomeric pore may contain any number of mutant monomers. Thepore typically comprises at least 7, at least 8, at least 9 or at least10 identical mutant monomers, such as 7, 8, 9 or 10 mutant monomers. Thepore preferably comprises eight identical mutant monomers. One or more,such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the mutant monomers ispreferably chemically modified as discussed above.

Methods for making pores are discussed in more detail below.

Hetero-Oligomeric Pores

The invention also provides a hetero-oligomeric pore derived from Mspcomprising at least one mutant monomer of the invention. Thehetero-oligomeric pore of the invention is ideal for characterising,such as sequencing, polynucleotides. Hetero-oligomeric pores can be madeusing methods known in the art (e.g. Protein Sci. 2002 July;11(7):1813-24).

The hetero-oligomeric pore contains sufficient monomers to form thepore. The monomers may be of any type. The pore typically comprises atleast 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9or 10 monomers. The pore preferably comprises eight monomers.

In a preferred embodiment, all of the monomers (such as 10, 9, 8 or 7 ofthe monomers) are mutant monomers of the invention and at least one ofthem differs from the others. In a more preferred embodiment, the porecomprises eight mutant monomers of the invention and at least one ofthem differs from the others. They may all differ from one another.

In another preferred embodiment, at least one of the mutant monomers isnot a mutant monomer of the invention. In this embodiment, the remainingmonomers are preferably mutant monomers of the invention. Hence, thepore may comprise 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutant monomers of theinvention. Any number of the monomers in the pore may not be a mutantmonomer of the invention. The pore preferably comprises seven mutantmonomers of the invention and a monomer which is not a monomer of theinvention, such as a monomer comprising a comparative variant asdiscussed above. The mutant monomers of the invention may be the same ordifferent.

In all the embodiments discussed above, one or more, such as 2, 3, 4, 5,6, 7, 8, 9 or 10, of the mutant monomers is preferably chemicallymodified as discussed above.

Methods for making pores are discussed in more detail below.

Construct-Containing Pores

The invention also provides a pore comprising at least one construct ofthe invention. A construct of the invention comprises two or morecovalently attached monomers derived from Msp wherein at least one ofthe monomers is a mutant monomer of the invention. In other words, aconstruct must contain more than one monomer. The pore containssufficient constructs and, if necessary, monomers to form the pore. Forinstance, an octameric pore may comprise (a) four constructs eachcomprising two constructs, (b) two constructs each comprising fourmonomers or (b) one construct comprising two monomers and six monomersthat do not form part of a construct. Other combinations of constructsand monomers can be envisaged by the skilled person.

At least two of the monomers in the pore are in the form of a constructof the invention. The construct, and hence the pore, comprises at leastone mutant monomer of the invention. The pore typically comprises atleast 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9or 10 monomers, in total (at least two of which must be in a construct).The pore preferably comprises eight monomers (at least two of which mustbe in a construct).

The construct containing pore may be a homo-oligomer (i.e. includeidentical constructs) or be a hetero-oligomer (i.e. where at least oneconstruct differs from the others).

A pore typically contains (a) one construct comprising two monomers and(b) 5, 6, 7 or 8 monomers. The construct may be any of those discussedabove. The monomers may be any of those discussed above, includingmutant monomers of the invention and mutant monomers comprising acomparative variant of SEQ ID NO: 2 as discussed above.

Another typical pore comprises more than one construct of the invention,such as two, three or four constructs of the invention. If necessary,such pores further comprise sufficient additional monomers or constructsto form the pore. The additional monomer(s) may be any of thosediscussed above, including mutant monomers of the invention and mutantmonomers comprising a comparative variant of SEQ ID NO: 2 as discussedabove. The additional construct(s) may be any of those discussed aboveor may be a construct comprising two or more covalently attached MspAmonomers each comprising a comparative variant of SEQ ID NO: 2 asdiscussed above.

A further pore of the invention comprises only constructs comprising 2monomers, for example a pore may comprise 4, 5, 6, 7 or 8 constructscomprising 2 monomers. At least one construct is a construct of theinvention, i.e. at least one monomer in the at least one construct, andpreferably each monomer in the at least one construct, is a mutantmonomer of the invention. All of the constructs comprising 2 monomersmay be constructs of the invention.

A specific pore according to the invention comprises four constructs ofthe invention each comprising two monomers, wherein at least one monomerin each construct, and preferably each monomer in each construct, is amutant monomer of the invention. The constructs may oligomerise into apore with a structure such that only one monomer of each constructcontributes to the channel of the pore. Typically the other monomers ofthe construct will be on the outside of the channel of the pore. Forexample, pores of the invention may comprise 5, 6, 7 or 8 constructscomprising 2 monomers where the channel comprises 8 monomers.

Mutations can be introduced into the construct as described above. Themutations may be alternating, i.e. the mutations are different for eachmonomer within a two monomer construct and the constructs are assembledas a homo-oligomer resulting in alternating modifications. In otherwords, monomers comprising MutA and MutB are fused and assembled to forman A-B:A-B:A-B:A-B pore. Alternatively, the mutations may beneighbouring, i.e. identical mutations are introduced into two monomersin a construct and this is then oligomerised with different mutantmonomers or constructs. In other words, monomers comprising MutA arefused follow by oligomerisation with MutB-containing monomers to formA-A:B:B:B:B:B:B.

One or more of the monomers of the invention in a construct-containingpore may be chemically-modified as discussed above.

Polynucleotide Characterisation

The invention provides a method of characterising a targetpolynucleotide. The method involves measuring one or morecharacteristics of the target polynucleotide. The target polynucleotidemay also be called the template polynucleotide or the polynucleotide ofinterest.

Polynucleotide

A polynucleotide, such as a nucleic acid, is a macromolecule comprisingtwo or more nucleotides. The polynucleotide or nucleic acid may compriseany combination of any nucleotides. The nucleotides can be naturallyoccurring or artificial. One or more nucleotides in the polynucleotidecan be oxidized or methylated. One or more nucleotides in thepolynucleotide may be damaged. For instance, the polynucleotide maycomprise a pyrimidine dimer. Such dimers are typically associated withdamage by ultraviolet light and are the primary cause of skin melanomas.One or more nucleotides in the polynucleotide may be modified, forinstance with a label or a tag. Suitable labels are described below. Thepolynucleotide may comprise one or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least onephosphate group. The nucleobase and sugar form a nucleoside.

The nucleobase is typically heterocyclic. Nucleobases include, but arenot limited to, purines and pyrimidines and more specifically adenine(A), guanine (G), thymine (T), uracil (U) and cytosine (C).

The sugar is typically a pentose sugar. Nucleotide sugars include, butare not limited to, ribose and deoxyribose. The sugar is preferably adeoxyribose.

The polynucleotide preferably comprises the following nucleosides:deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT),deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotide is typically a ribonucleotide or deoxyribonucleotide. Thenucleotide typically contains a monophosphate, diphosphate ortriphosphate. The nucleotide may comprise more than three phosphates,such as 4 or 5 phosphates. Phosphates may be attached on the 5′ or 3′side of a nucleotide. Nucleotides include, but are not limited to,adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidinemonophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidinemonophosphate, 5-hydroxymethylcytidine monophosphate, cytidinemonophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclicguanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate(dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate(dCMP) and deoxymethylcytidine monophosphate. The nucleotides arepreferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMPand dUMP.

A nucleotide may be abasic (i.e. lack a nucleobase). A nucleotide mayalso lack a nucleobase and a sugar (i.e. is a C3 spacer).

The nucleotides in the polynucleotide may be attached to each other inany manner. The nucleotides are typically attached by their sugar andphosphate groups as in nucleic acids. The nucleotides may be connectedvia their nucleobases as in pyrimidine dimers.

The polynucleotide may be single stranded or double stranded. At least aportion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The polynucleotide can comprise onestrand of RNA hybridised to one strand of DNA. The polynucleotide may beany synthetic nucleic acid known in the art, such as peptide nucleicacid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA),locked nucleic acid (LNA) or other synthetic polymers with nucleotideside chains. The PNA backbone is composed of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backboneis composed of repeating glycol units linked by phosphodiester bonds.The TNA backbone is composed of repeating threose sugars linked togetherby phosphodiester bonds. LNA is formed from ribonucleotides as discussedabove having an extra bridge connecting the 2′ oxygen and 4′ carbon inthe ribose moiety.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) ordeoxyribonucleic acid (DNA).

The polynucleotide can be any length. For example, the polynucleotidecan be at least 10, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 400 or at least 500nucleotides or nucleotide pairs in length. The polynucleotide can be1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotidesor nucleotide pairs in length or 100000 or more nucleotides ornucleotide pairs in length.

Any number of polynucleotides can be investigated. For instance, themethod of the invention may concern characterising 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 50, 100 or more polynucleotides. If two or morepolynucleotides are characterized, they may be different polynucleotidesor two instances of the same polynucleotide.

The polynucleotide can be naturally occurring or artificial. Forinstance, the method may be used to verify the sequence of amanufactured oligonucleotide. The method is typically carried out invitro.

Sample

Each analyte is typically present in any suitable sample. The inventionis typically carried out on two or more samples that are known tocontain or suspected to contain the analytes. Alternatively, theinvention may be carried out on two or more samples to confirm theidentity of two or more analytes whose presence in the samples is knownor expected.

The first sample and/or second sample may be a biological sample. Theinvention may be carried out in vitro using at least one sample obtainedfrom or extracted from any organism or microorganism. The first sampleand/or second sample may be a non-biological sample. The non-biologicalsample is preferably a fluid sample. Examples of non-biological samplesinclude surgical fluids, water such as drinking water, sea water orriver water, and reagents for laboratory tests.

The first sample and/or second sample is typically processed prior tobeing used in the invention, for example by centrifugation or by passagethrough a membrane that filters out unwanted molecules or cells, such asred blood cells. The first sample and/or second sample may be measuredimmediately upon being taken. The first sample and/or second sample mayalso be typically stored prior to assay, preferably below −70° C.

Characterisation

The method may involve measuring two, three, four or five or morecharacteristics of the polynucleotide. The one or more characteristicsare preferably selected from (i) the length of the polynucleotide, (ii)the identity of the polynucleotide, (iii) the sequence of thepolynucleotide, (iv) the secondary structure of the polynucleotide and(v) whether or not the polynucleotide is modified. Any combination of(i) to (v) may be measured in accordance with the invention, such as{i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii},{ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iv}, {i,ii,v},{i,iii,iv}, {i,iii,v}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v}, {ii,iv,v},{iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v}, {i,iii,iv,v},{ii,iii,iv,v} or {i,ii,iii,iv,v}. Different combinations of (i) to (v)may be measured for the first polynucleotide compared with the secondpolynucleotide, including any of those combinations listed above.

For (i), the length of the polynucleotide may be measured for example bydetermining the number of interactions between the polynucleotide andthe pore or the duration of interaction between the polynucleotide andthe pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the polynucleotide orwithout measurement of the sequence of the polynucleotide. The former isstraightforward; the polynucleotide is sequenced and thereby identified.The latter may be done in several ways. For instance, the presence of aparticular motif in the polynucleotide may be measured (withoutmeasuring the remaining sequence of the polynucleotide). Alternatively,the measurement of a particular electrical and/or optical signal in themethod may identify the polynucleotide as coming from a particularsource.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc.2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not thepolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcyotsine may be distinguished from cytosine on the basisof the current flowing through the pore during its interaction with eachnucleotide.

The target polynucleotide is contacted with a pore of the invention. Thepore is typically present in a membrane. Suitable membranes arediscussed below. The method may be carried out using any apparatus thatis suitable for investigating a membrane/pore system in which a pore ispresent in a membrane. The method may be carried out using any apparatusthat is suitable for transmembrane pore sensing. For example, theapparatus comprises a chamber comprising an aqueous solution and abarrier that separates the chamber into two sections. The barriertypically has an aperture in which the membrane containing the pore isformed. Alternatively the barrier forms the membrane in which the poreis present.

The method may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562 (WO 2008/102120).

A variety of different types of measurements may be made. This includeswithout limitation: electrical measurements and optical measurements.Possible electrical measurements include: current measurements,impedance measurements, tunnelling measurements (Ivanov A P et al., NanoLett. 2011 Jan. 12; 11(1):279-85), and FET measurements (InternationalApplication WO 2005/124888). Optical measurements may be combined withelectrical measurements (Soni G V et al., Rev Sci Instrum. 2010 January;81(1):014301). The measurement may be a transmembrane currentmeasurement such as measurement of ionic current flowing through thepore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010;132(50):17961-72, and International Application WO 2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International Application WO2009/077734 and International Application WO 2011/067559.

The method is preferably carried out with a potential applied across themembrane. The applied potential may be a voltage potential.Alternatively, the applied potential may be a chemical potential. Anexample of this is using a salt gradient across a membrane, such as anamphiphilic layer. A salt gradient is disclosed in Holden et al., J AmChem Soc. 2007 Jul. 11; 129(27):8650-5. In some instances, the currentpassing through the pore as a polynucleotide moves with respect to thepore is used to estimate or determine the sequence of thepolynucleotide. This is strand sequencing.

The method may involve measuring the current passing through the pore asthe polynucleotide moves with respect to the pore. Therefore theapparatus used in the method may also comprise an electrical circuitcapable of applying a potential and measuring an electrical signalacross the membrane and pore. The methods may be carried out using apatch clamp or a voltage clamp. The methods preferably involve the useof a voltage clamp.

The method of the invention may involve the measuring of a currentpassing through the pore as the polynucleotide moves with respect to thepore. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExample. The method is typically carried out with a voltage appliedacross the membrane and pore. The voltage used is typically from +5 V to−5 V, such as from +4 V to −4 V, +3 V to −3 V or +2 V to −2 V. Thevoltage used is typically from −600 mV to +600 mV or −400 mV to +400 mV.The voltage used is preferably in a range having a lower limit selectedfrom −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0mV and an upper limit independently selected from +10 mV, +20 mV, +50mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used ismore preferably in the range 100 mV to 240 mV and most preferably in therange of 120 mV to 220 mV. It is possible to increase discriminationbetween different nucleotides by a pore by using an increased appliedpotential.

The method is typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture ofpotassium ferrocyanide and potassium ferricyanide is typically used.KCl, NaCl and a mixture of potassium ferrocyanide and potassiumferricyanide are preferred. The charge carriers may be asymmetric acrossthe membrane. For instance, the type and/or concentration of the chargecarriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration maybe 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M,from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to1.4 M. The salt concentration is preferably from 150 mM to 1 M. Themethod is preferably carried out using a salt concentration of at least0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M orat least 3.0 M. High salt concentrations provide a high signal to noiseratio and allow for currents indicative of the presence of a nucleotideto be identified against the background of normal current fluctuations.

The method is typically carried out in the presence of a buffer. In theexemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is phosphate buffer. Other suitablebuffers are HEPES and Tris-HCl buffer. The methods are typically carriedout at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pHused is preferably about 7.5.

The method may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

Polynucleotide Binding Protein

Step (a) preferably comprises contacting the polynucleotide with apolynucleotide binding protein such that the protein controls themovement of the polynucleotide through the pore.

More preferably, the method comprises (a) contacting the polynucleotidewith the pore of the invention and a polynucleotide binding protein suchthat the protein controls the movement of the polynucleotide through thepore and (b) taking one or more measurements as the polynucleotide moveswith respect to the pore, wherein the measurements are indicative of oneor more characteristics of the polynucleotide, and therebycharacterising the polynucleotide.

More preferably, the method comprises (a) contacting the polynucleotidewith the pore of the invention and a polynucleotide binding protein suchthat the protein controls the movement of the polynucleotide through thepore and (b) measuring the current through the pore as thepolynucleotide moves with respect to the pore, wherein the current isindicative of one or more characteristics of the polynucleotide, andthereby characterising the polynucleotide.

The polynucleotide binding protein may be any protein that is capable ofbinding to the polynucleotide and controlling its movement through thepore. It is straightforward in the art to determine whether or not aprotein binds to a polynucleotide. The protein typically interacts withand modifies at least one property of the polynucleotide. The proteinmay modify the polynucleotide by cleaving it to form individualnucleotides or shorter chains of nucleotides, such as di- ortrinucleotides. The protein may modify the polynucleotide by orientingit or moving it to a specific position, i.e. controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. A polynucleotide handling enzyme is apolypeptide that is capable of interacting with and modifying at leastone property of a polynucleotide. The enzyme may modify thepolynucleotide by cleaving it to form individual nucleotides or shorterchains of nucleotides, such as di- or trinucleotides. The enzyme maymodify the polynucleotide by orienting it or moving it to a specificposition. The polynucleotide handling enzyme does not need to displayenzymatic activity as long as it is capable of binding thepolynucleotide and controlling its movement through the pore. Forinstance, the enzyme may be modified to remove its enzymatic activity ormay be used under conditions which prevent it from acting as an enzyme.Such conditions are discussed in more detail below.

The polynucleotide handling enzyme is preferably derived from anucleolytic enzyme. The polynucleotide handling enzyme used in theconstruct of the enzyme is more preferably derived from a member of anyof the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. Theenzyme may be any of those disclosed in International Application No.PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases andtopoisomerases, such as gyrases. Suitable enzymes include, but are notlimited to, exonuclease I from E. coli (SEQ ID NO: 11), exonuclease IIIenzyme from E. coli (SEQ ID NO: 13), RecJ from T. thermophilus (SEQ IDNO: 15) and bacteriophage lambda exonuclease (SEQ ID NO: 17) andvariants thereof. Three subunits comprising the sequence shown in SEQ IDNO: 15 or a variant thereof interact to form a trimer exonuclease. Theenzyme is preferably Phi29 DNA polymerase (SEQ ID NO: 9) or a variantthereof. The topoisomerase is preferably a member of any of the MoietyClassification (EC) groups 5.99.1.2 and 5.99.1.3.

The enzyme is most preferably derived from a helicase, such as Hel308Mbu (SEQ ID NO: 18), Hel308 Csy (SEQ ID NO: 19), Hel308 Mhu (SEQ ID NO:21), TraI Eco (SEQ ID NO: 22), XPD Mbu (SEQ ID NO: 23) or a variantthereof. Any helicase may be used in the invention. The helicase may beor be derived from a Hel308 helicase, a RecD helicase, such as TraIhelicase or a TrwC helicase, a XPD helicase or a Dda helicase. Thehelicase may be any of the helicases, modified helicases or helicaseconstructs disclosed in International Application Nos. PCT/GB2012/052579(published as WO 2013/057495); PCT/GB2012/053274 (published as WO2013/098562); PCT/GB2012/053273 (published as WO2013098561);PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924(published as WO 2014/013259) and PCT/GB2013/051928 (published as WO2014/013262); and in PCT/GB2014/052736 (published as WO/2015/055981).

The helicase preferably comprises the sequence shown in SEQ ID NO: 25(Trwc Cba) or as variant thereof, the sequence shown in SEQ ID NO: 18(Hel308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 24(Dda) or a variant thereof. Variants may differ from the nativesequences in any of the ways discussed below for transmembrane pores. Apreferred variant of SEQ ID NO: 8 comprises E94C/A360C and then(ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and G2).

Any number of helicases may be used in accordance with the invention.For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may beused. In some embodiments, different numbers of helicases may be used.

The method of the invention preferably comprises contacting thepolynucleotide with two or more helicases. The two or more helicases aretypically the same helicase. The two or more helicases may be differenthelicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. Preferred helicase constructs for use in the invention aredescribed in International Application Nos. PCT/GB2013/051925 (publishedas WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259) andPCT/GB2013/051928 (published as WO 2014/013262); and inPCT/GB2014/052736 (published as WO/2015/055981).

A variant of SEQ ID NOs: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24or 25 is an enzyme that has an amino acid sequence which varies fromthat of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25and which retains polynucleotide binding ability. This can be measuredusing any method known in the art. For instance, the variant can becontacted with a polynucleotide and its ability to bind to and movealong the polynucleotide can be measured. The variant may includemodifications that facilitate binding of the polynucleotide and/orfacilitate its activity at high salt concentrations and/or roomtemperature. Variants may be modified such that they bindpolynucleotides (i.e. retain polynucleotide binding ability) but do notfunction as a helicase (i.e. do not move along polynucleotides whenprovided with all the necessary components to facilitate movement, e.g.ATP and Mg²⁺). Such modifications are known in the art. For instance,modification of the Mg²⁺ binding domain in helicases typically resultsin variants which do not function as helicases. These types of variantsmay act as molecular brakes (see below).

Over the entire length of the amino acid sequence of SEQ ID NO: 9, 11,13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25, a variant will preferablybe at least 50% homologous to that sequence based on amino acididentity. More preferably, the variant polypeptide may be at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% and more preferably at least 95%, 97% or 99%homologous based on amino acid identity to the amino acid sequence ofSEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95%, amino acid identity over a stretch of 200 or more, forexample 230, 250, 270, 280, 300, 400, 500, 600, 700, 800, 900 or 1000 ormore, contiguous amino acids (“hard homology”). Homology is determinedas described above. The variant may differ from the wild-type sequencein any of the ways discussed above with reference to SEQ ID NO: 2 and 4above. The enzyme may be covalently attached to the pore. Any method maybe used to covalently attach the enzyme to the pore.

A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 25 with themutation Q594A). This variant does not function as a helicase (i.e.binds polynucleotides but does not move along them when provided withall the necessary components to facilitate movement, e.g. ATP and Mg²⁺).

In strand sequencing, the polynucleotide is translocated through thepore either with or against an applied potential. Exonucleases that actprogressively or processively on double stranded polynucleotides can beused on the cis side of the pore to feed the remaining single strandthrough under an applied potential or the trans side under a reversepotential. Likewise, a helicase that unwinds the double stranded DNA canalso be used in a similar manner. A polymerase may also be used. Thereare also possibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

Any helicase may be used in the method. Helicases may work in two modeswith respect to the pore. First, the method is preferably carried outusing a helicase such that it moves the polynucleotide through the porewith the field resulting from the applied voltage. In this mode the 5′end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide into the pore such that it is passedthrough the pore with the field until it finally translocates through tothe trans side of the membrane. Alternatively, the method is preferablycarried out such that a helicase moves the polynucleotide through thepore against the field resulting from the applied voltage. In this modethe 3′ end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide through the pore such that it ispulled out of the pore against the applied field until finally ejectedback to the cis side of the membrane.

The method may also be carried out in the opposite direction. The 3′ endof the polynucleotide may be first captured in the pore and the helicasemay move the polynucleotide into the pore such that it is passed throughthe pore with the field until it finally translocates through to thetrans side of the membrane.

When the helicase is not provided with the necessary components tofacilitate movement or is modified to hinder or prevent its movement, itcan bind to the polynucleotide and act as a brake slowing the movementof the polynucleotide when it is pulled into the pore by the appliedfield. In the inactive mode, it does not matter whether thepolynucleotide is captured either 3′ or 5′ down, it is the applied fieldwhich pulls the polynucleotide into the pore towards the trans side withthe enzyme acting as a brake. When in the inactive mode, the movementcontrol of the polynucleotide by the helicase can be described in anumber of ways including ratcheting, sliding and braking. Helicasevariants which lack helicase activity can also be used in this way.

The polynucleotide may be contacted with the polynucleotide bindingprotein and the pore in any order. It is preferred that, when thepolynucleotide is contacted with the polynucleotide binding protein,such as a helicase, and the pore, the polynucleotide firstly forms acomplex with the protein. When the voltage is applied across the pore,the polynucleotide/protein complex then forms a complex with the poreand controls the movement of the polynucleotide through the pore.

Any steps in the method using a polynucleotide binding protein aretypically carried out in the presence of free nucleotides or freenucleotide analogues and an enzyme cofactor that facilitates the actionof the polynucleotide binding protein. The free nucleotides may be oneor more of any of the individual nucleotides discussed above. The freenucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The free nucleotides are preferably selected from AMP, TMP, GMP, CMP,UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferablyadenosine triphosphate (ATP). The enzyme cofactor is a factor thatallows the construct to function. The enzyme cofactor is preferably adivalent metal cation. The divalent metal cation is preferably Mg²⁺,Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactor is most preferably Mg²⁺.

Helicase(s) and Molecular Brake(s)

In a preferred embodiment, the method comprises:

-   -   (a) providing the polynucleotide with one or more helicases and        one or more molecular brakes attached to the polynucleotide;    -   (b) contacting the polynucleotide with a pore of the invention        and applying a potential across the pore such that the one or        more helicases and the one or more molecular brakes are brought        together and both control the movement of the polynucleotide        through the pore;    -   (c) taking one or more measurements as the polynucleotide moves        with respect to the pore wherein the measurements are indicative        of one or more characteristics of the polynucleotide and thereby        characterising the polynucleotide. This type of method is        discussed in detail in International Application No.        PCT/GB2014/052737.

The one or more helicases may be any of those discussed above. The oneor more molecular brakes may be any compound or molecule which binds tothe polynucleotide and slow the movement of the polynucleotide throughthe pore. The one or more molecular brakes preferably comprise one ormore compounds which bind to the polynucleotide. The one or morecompounds are preferably one or more macrocycles. Suitable macrocyclesinclude, but are not limited to, cyclodextrins, calixarenes, cyclicpeptides, crown ethers, cucurbiturils, pillararenes, derivatives thereofor a combination thereof. The cyclodextrin or derivative thereof may beany of those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J.Am. Chem. Soc. 116, 6081-6088. The agent is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD).

The one or more molecular brakes are preferably not one or more singlestranded binding proteins (SSB). The one or more molecular brakes aremore preferably not a single-stranded binding protein (SSB) comprising acarboxy-terminal (C-terminal) region which does not have a net negativecharge or (ii) a modified SSB comprising one or more modifications inits C-terminal region which decreases the net negative charge of theC-terminal region. The one or more molecular brakes are most preferablynot any of the SSBs disclosed in International Application No.PCT/GB2013/051924 (published as WO 2014/013259).

The one or more molecular brakes are preferably one or morepolynucleotide binding proteins. The polynucleotide binding protein maybe any protein that is capable of binding to the polynucleotide andcontrolling its movement through the pore. It is straightforward in theart to determine whether or not a protein binds to a polynucleotide. Theprotein typically interacts with and modifies at least one property ofthe polynucleotide. The protein may modify the polynucleotide bycleaving it to form individual nucleotides or shorter chains ofnucleotides, such as di- or trinucleotides. The moiety may modify thepolynucleotide by orienting it or moving it to a specific position, i.e.controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. The one or more molecular brakes may bederived from any of the polynucleotide handling enzymes discussed above.Modified versions of Phi29 polymerase (SEQ ID NO: 8) which act asmolecular brakes are disclosed in U.S. Pat. No. 5,576,204. The one ormore molecular brakes are preferably derived from a helicase.

Any number of molecular brakes derived from a helicase may be used. Forinstance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may be used asmolecular brakes. If two or more helicases are be used as molecularbrakes, the two or more helicases are typically the same helicase. Thetwo or more helicases may be different helicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. The one or more molecular brakes derived from helicases arepreferably modified to reduce the size of an opening in thepolynucleotide binding domain through which in at least oneconformational state the polynucleotide can unbind from the helicase.This is disclosed in WO 2014/013260.

Preferred helicase constructs for use in the invention are described inInternational Application Nos. PCT/GB2013/051925 (published as WO2014/013260); PCT/GB2013/051924 (published as WO 2014/013259) andPCT/GB2013/051928 (published as WO 2014/013262); and inPCT/GB2014/052736 (published as WO/2015/055981).

Spacers

The one or more helicases may be stalled at one or more spacers asdiscussed in International Application No. PCT/GB2014/050175. Anyconfiguration of one or more helicases and one or more spacers disclosedin the International Application may be used in this invention.

Membrane

The pore of the invention may be present in a membrane. In the method ofthe invention, the polynucleotide is typically contacted with the poreof the invention in a membrane. Any membrane may be used in accordancewith the invention. Suitable membranes are well-known in the art. Themembrane is preferably an amphiphilic layer. An amphiphilic layer is alayer formed from amphiphilic molecules, such as phospholipids, whichhave both hydrophilic and lipophilic properties. The amphiphilicmolecules may be synthetic or naturally occurring. Non-naturallyoccurring amphiphiles and amphiphiles which form a monolayer are knownin the art and include, for example, block copolymers (Gonzalez-Perez etal., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymericmaterials in which two or more monomer sub-units that are polymerizedtogether to create a single polymer chain. Block copolymers typicallyhave properties that are contributed by each monomer sub-unit. However,a block copolymer may have unique properties that polymers formed fromthe individual sub-units do not possess. Block copolymers can beengineered such that one of the monomer sub-units is hydrophobic (i.e.lipophilic), whilst the other sub-unit(s) are hydrophilic whilst inaqueous media. In this case, the block copolymer may possess amphiphilicproperties and may form a structure that mimics a biological membrane.The block copolymer may be a diblock (consisting of two monomersub-units), but may also be constructed from more than two monomersub-units to form more complex arrangements that behave as amphipiles.The copolymer may be a triblock, tetrablock or pentablock copolymer. Themembrane is preferably a triblock copolymer membrane.

Archaebacterial bipolar tetraether lipids are naturally occurring lipidsthat are constructed such that the lipid forms a monolayer membrane.These lipids are generally found in extremophiles that survive in harshbiological environments, thermophiles, halophiles and acidophiles. Theirstability is believed to derive from the fused nature of the finalbilayer. It is straightforward to construct block copolymer materialsthat mimic these biological entities by creating a triblock polymer thathas the general motif hydrophilic-hydrophobic-hydrophilic. This materialmay form monomeric membranes that behave similarly to lipid bilayers andencompass a range of phase behaviours from vesicles through to laminarmembranes. Membranes formed from these triblock copolymers hold severaladvantages over biological lipid membranes. Because the triblockcopolymer is synthesized, the exact construction can be carefullycontrolled to provide the correct chain lengths and properties requiredto form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are notclassed as lipid sub-materials; for example a hydrophobic polymer may bemade from siloxane or other non-hydrocarbon based monomers. Thehydrophilic sub-section of block copolymer can also possess low proteinbinding properties, which allows the creation of a membrane that ishighly resistant when exposed to raw biological samples. This head groupunit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical andenvironmental stability compared with biological lipid membranes, forexample a much higher operational temperature or pH range. The syntheticnature of the block copolymers provides a platform to customize polymerbased membranes for a wide range of applications.

The membrane is most preferably one of the membranes disclosed inInternational Application No. PCT/GB2013/052766 or PCT/GB2013/052767.

The amphiphilic molecules may be chemically-modified or functionalisedto facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically planar. The amphiphilic layer may be curved. Theamphiphilic layer may be supported.

Amphiphilic membranes are typically naturally mobile, essentially actingas two dimensional fluids with lipid diffusion rates of approximately10⁸ cm s-1. This means that the pore and coupled polynucleotide cantypically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cellmembranes and serve as excellent platforms for a range of experimentalstudies. For example, lipid bilayers can be used for in vitroinvestigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Coupling

The polynucleotide is preferably coupled to the membrane comprising thepore of the invention. The method may comprise coupling thepolynucleotide to the membrane comprising the pore of the invention. Thepolynucleotide is preferably coupled to the membrane using one or moreanchors. The polynucleotide may be coupled to the membrane using anyknown method.

Each anchor comprises a group which couples (or binds) to thepolynucleotide and a group which couples (or binds) to the membrane.Each anchor may covalently couple (or bind) to the polynucleotide and/orthe membrane. If a Y adaptor and/or a hairpin loop adaptors are used,the polynucleotide is preferably coupled to the membrane using theadaptor(s).

The polynucleotide may be coupled to the membrane using any number ofanchors, such as 2, 3, 4 or more anchors. For instance, a polynucleotidemay be coupled to the membrane using two anchors each of whichseparately couples (or binds) to both the polynucleotide and membrane.

The one or more anchors may comprise the one or more helicases and/orthe one or more molecular brakes.

If the membrane is an amphiphilic layer, such as a copolymer membrane ora lipid bilayer, the one or more anchors preferably comprise apolypeptide anchor present in the membrane and/or a hydrophobic anchorpresent in the membrane. The hydrophobic anchor is preferably a lipid,fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid,for example cholesterol, palmitate or tocopherol. In preferredembodiments, the one or more anchors are not the pore.

The components of the membrane, such as the amphiphilic molecules,copolymer or lipids, may be chemically-modified or functionalised toform the one or more anchors. Examples of suitable chemicalmodifications and suitable ways of functionalising the components of themembrane are discussed in more detail below. Any proportion of themembrane components may be functionalized, for example at least 0.01%,at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or100%.

The polynucleotide may be coupled directly to the membrane. The one ormore anchors used to couple the polynucleotide to the membranepreferably comprise a linker. The one or more anchors may comprise oneor more, such as 2, 3, 4 or more, linkers. One linker may be used couplemore than one, such as 2, 3, 4 or more, polynucleotides to the membrane.

Preferred linkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs), polysaccharides andpolypeptides. These linkers may be linear, branched or circular. Forinstance, the linker may be a circular polynucleotide. Thepolynucleotide may hybridise to a complementary sequence on the circularpolynucleotide linker.

The coupling may be permanent or stable. In other words, the couplingmay be such that the polynucleotide remains coupled to the membrane wheninteracting with the pore.

The coupling may be transient. In other words, the coupling may be suchthat the polynucleotide may decouple from the membrane when interactingwith the pore.

Coupling of polynucleotides to a linker or to a functionalised membranecan also be achieved by a number of other means provided that acomplementary reactive group or an anchoring group can be added to thepolynucleotide. The addition of reactive groups to either end of apolynucleotide has been reported previously. The one or more anchorspreferably couple the polynucleotide to the membrane via hybridisation.Hybridisation in the one or more anchors allows coupling in a transientmanner as discussed above. The one or more anchors may comprise a singlestranded or double stranded polynucleotide. One part of the anchor maybe ligated to a single stranded or double stranded polynucleotide.Ligation of short pieces of ssDNA have been reported using T4 RNA ligaseI (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).“Ligation-anchored PCR: a simple amplification technique withsingle-sided specificity.” Proc Natl Acad Sci USA 89(20): 9823-5).Alternatively, either a single stranded or double strandedpolynucleotide can be ligated to a double stranded polynucleotide andthen the two strands separated by thermal or chemical denaturation. Ifthe polynucleotide is a synthetic strand, the one or more anchors can beincorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesised using a primer having areactive group attached to it.

Ideally, the polynucleotide is coupled to the membrane without having tofunctionalise the polynucleotide. This can be achieved by coupling theone or more anchors, such as a polynucleotide binding protein or achemical group, to the membrane and allowing the one or more anchors tointeract with the polynucleotide or by functionalizing the membrane. Theone or more anchors may be coupled to the membrane by any of the methodsdescribed herein. In particular, the one or more anchors may compriseone or more linkers, such as maleimide functionalised linkers.

In this embodiment, the polynucleotide is typically RNA, DNA, PNA, TNAor LNA and may be double or single stranded. This embodiment isparticularly suited to genomic DNA polynucleotides.

Where the one or more anchors comprise a protein, they may be able toanchor directly into the membrane without further functonalisation, forexample if it already has an external hydrophobic region which iscompatible with the membrane. Examples of such proteins include, but arenot limited to, transmembrane proteins, intramembrane proteins andmembrane proteins.

According to a preferred embodiment, the one or more anchors may be usedto couple a polynucleotide to the membrane when the polynucleotide isattached to a leader sequence which preferentially threads into thepore. Leader sequences are discussed in more detail below. Preferably,the polynucleotide is attached (such as ligated) to a leader sequencewhich preferentially threads into the pore. Such a leader sequence maycomprise a homopolymeric polynucleotide or an abasic region. The leadersequence is typically designed to hybridise to the one or more anchorseither directly or via one or more intermediate polynucleotides (orsplints). In such instances, the one or more anchors typically comprisea polynucleotide sequence which is complementary to a sequence in theleader sequence or a sequence in the one or more intermediatepolynucleotides (or splints). In such instances, the one or more splintstypically comprise a polynucleotide sequence which is complementary to asequence in the leader sequence.

Double Stranded Polynucleotide

The polynucleotide may be double stranded. If the polynucleotide isdouble stranded, the method preferably further comprises before thecontacting step ligating a hairpin adaptor to one end of thepolynucleotide. The two strands of the polynucleotide may then beseparated as or before the polynucleotide is contacted with the pore inaccordance with the invention. The two strands may be separated as thepolynucleotide movement through the pore is controlled by apolynucleotide binding protein, such as a helicase, or molecular brake.This is described in International Application No. PCT/GB2012/051786(published as WO 2013/014451). Linking and interrogating both strands ona double stranded construct in this way increases the efficiency andaccuracy of characterization.

Round the Corner Sequencing

In a preferred embodiment, a target double stranded polynucleotide isprovided with a hairpin loop adaptor at one end and the method comprisescontacting the polynucleotide with the pore of the invention such thatboth strands of the polynucleotide move through the pore and taking oneor more measurements as the both strands of the polynucleotide move withrespect to the pore wherein the measurements are indicative of one ormore characteristics of the strands of the polynucleotide and therebycharacterising the target double stranded polynucleotide. Any of theembodiments discussed above equally apply to this embodiment.

Leader Sequence

Before the contacting step, the method preferably comprises attaching tothe polynucleotide a leader sequence which preferentially threads intothe pore. The leader sequence facilitates the method of the invention.The leader sequence is designed to preferentially thread into the poreof the invention and thereby facilitate the movement of polynucleotidethrough the pore. The leader sequence can also be used to link thepolynucleotide to the one or more anchors as discussed above.

Double Coupling

The method of the invention may involve double coupling of a doublestranded polynucleotide. In a preferred embodiment, the method of theinvention comprises:

(a) providing the double stranded polynucleotide with a Y adaptor at oneend and a hairpin loop adaptor at the other end, wherein the Y adaptorcomprises one or more first anchors for coupling the polynucleotide tothe membrane, wherein the hairpin loop adaptor comprises one or moresecond anchors for coupling the polynucleotide to the membrane andwherein the strength of coupling of the hairpin loop adaptor to themembrane is greater than the strength of coupling of the Y adaptor tothe membrane;

(b) contacting the polynucleotide provided in step (a) with the pore theinvention such that the polynucleotide moves through the pore; and

(c) taking one or more measurements as the polynucleotide moves withrespect to the pore, wherein the measurements are indicative of one ormore characteristics of the polynucleotide, and thereby characterisingthe target polynucleotide. This type of method is discussed in detail inInternational Application No. PCT/GB2015/050991.

Adding Hairpin Loops and Leader Sequences

Before provision, a double stranded polynucleotide may be contacted witha MuA transposase and a population of double stranded MuA substrates,wherein a proportion of the substrates in the population are Y adaptorscomprising the leader sequence and wherein a proportion of thesubstrates in the population are hairpin loop adaptors. The transposasefragments the double stranded polynucleotide analyte and ligates MuAsubstrates to one or both ends of the fragments. This produces aplurality of modified double stranded polynucleotides comprising theleader sequence at one end and the hairpin loop at the other. Themodified double stranded polynucleotides may then be investigated usingthe method of the invention. These MuA based methods are disclosed inInternational Application No. PCT/GB2014/052505 (published asWO/2015/022544). They are also discussed in detail in InternationalApplication No. PCT/GB2015/050991.

One or more helicases may be attached to the MuA substrate Y adaptorsbefore they are contacted with the double stranded polynucleotide andMuA transposase. Alternatively, one or more helicases may be attached tothe MuA substrate Y adaptors before they are contacted with the doublestranded polynucleotide and MuA transposase.

One or more molecular brakes may be attached to the MuA substratehairpin loop adaptors before they are contacted with the double strandedpolynucleotide and MuA transposase. Alternatively, one or more molecularbrakes may be attached to the MuA substrate hairpin loop adaptors beforethey are contacted with the double stranded polynucleotide and MuAtransposase.

Uncoupling

The method of the invention may involve characterising multiple targetpolynucleotides and uncoupling of the at least the first targetpolynucleotide.

In a preferred embodiment, the invention involves characterising two ormore target polynucleotides. The method comprises:

-   -   (a) providing a first polynucleotide in a first sample;    -   (b) providing a second polynucleotide in a second sample;    -   (c) coupling the first polynucleotide in the first sample to a        membrane using one or more anchors;    -   (d) contacting the first polynucleotide with the pore of the        invention such that the polynucleotide moves through the pore;    -   (e) taking one or more measurements as the first polynucleotide        moves with respect to the pore wherein the measurements are        indicative of one or more characteristics of the first        polynucleotide and thereby characterising the first        polynucleotide;    -   (f) uncoupling the first polynucleotide from the membrane;    -   (g) coupling the second polynucleotide in the second sample to        the membrane using one or more anchors;    -   (h) contacting the second polynucleotide with the pore of the        invention such that the second polynucleotide moves through the        pore; and    -   (i) taking one or more measurements as the second polynucleotide        moves with respect to the pore wherein the measurements are        indicative of one or more characteristics of the second        polynucleotide and thereby characterising the second        polynucleotide.

This type of method is discussed in detail in International ApplicationNo. PCT/GB2015/050992.

If one or more anchors comprise a hydrophobic anchor, such ascholesterol, the agent is preferably a cyclodextrin or a derivativethereof or a lipid. The cyclodextrin or derivative thereof may be any ofthose disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am.Chem. Soc. 116, 6081-6088. The agent is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). Any of the lipidsdisclosed herein may be used.

Modified Polynucleotides

Before characterisation, a target polynucleotide may be modified bycontacting the polynucleotide with a polymerase and a population of freenucleotides under conditions in which the polymerase forms a modifiedpolynucleotide using the target polynucleotide as a template, whereinthe polymerase replaces one or more of the nucleotide species in thetarget polynucleotide with a different nucleotide species when formingthe modified polynucleotide. The modified polynucleotide may then beprovided with one or more helicases attached to the polynucleotide andone or more molecular brakes attached to the polynucleotide. This typeof modification is described in International Application No.PCT/GB2015/050483. Any of the polymerases discussed above may be used.The polymerase is preferably Klenow or 90 North.

The template polynucleotide is contacted with the polymerase underconditions in which the polymerase forms a modified polynucleotide usingthe template polynucleotide as a template. Such conditions are known inthe art. For instance, the polynucleotide is typically contacted withthe polymerase in commercially available polymerase buffer, such asbuffer from New England Biolabs®. The temperature is preferably from 20to 37° C. for Klenow or from 60 to 75° C. for 90 North. A primer or a 3′hairpin is typically used as the nucleation point for polymeraseextension.

Characterisation, such as sequencing, of a polynucleotide using atransmembrane pore typically involves analyzing polymer units made up ofk nucleotides where k is a positive integer (i.e. ‘k-mers’). This isdiscussed in International Application No. PCT/GB2012/052343 (publishedas WO 2013/041878). While it is desirable to have clear separationbetween current measurements for different k-mers, it is common for someof these measurements to overlap. Especially with high numbers ofpolymer units in the k-mer, i.e. high values of k, it can becomedifficult to resolve the measurements produced by different k-mers, tothe detriment of deriving information about the polynucleotide, forexample an estimate of the underlying sequence of the polynucleotide.

By replacing one or more nucleotide species in the target polynucleotidewith different nucleotide species in the modified polynucleotide, themodified polynucleotide contains k-mers which differ from those in thetarget polynucleotide. The different k-mers in the modifiedpolynucleotide are capable of producing different current measurementsfrom the k-mers in the target polynucleotide and so the modifiedpolynucleotide provides different information from the targetpolynucleotide. The additional information from the modifiedpolynucleotide can make it easier to characterise the targetpolynucleotide. In some instances, the modified polynucleotide itselfmay be easier to characterise. For instance, the modified polynucleotidemay be designed to include k-mers with an increased separation or aclear separation between their current measurements or k-mers which havea decreased noise.

The polymerase preferably replaces two or more of the nucleotide speciesin the target polynucleotide with different nucleotide species whenforming the modified polynucleotide. The polymerase may replace each ofthe two or more nucleotide species in the target polynucleotide with adistinct nucleotide species. The polymerase may replace each of the twoor more nucleotide species in the target polynucleotide with the samenucleotide species.

If the target polynucleotide is DNA, the different nucleotide species inthe modified typically comprises a nucleobase which differs fromadenine, guanine, thymine, cytosine or methylcytosine and/or comprises anucleoside which differs from deoxyadenosine, deoxyguanosine, thymidine,deoxycytidine or deoxymethylcytidine. If the target polynucleotide isRNA, the different nucleotide species in the modified polynucleotidetypically comprises a nucleobase which differs from adenine, guanine,uracil, cytosine or methylcytosine and/or comprises a nucleoside whichdiffers from adenosine, guanosine, uridine, cytidine or methylcytidine.The different nucleotide species may be any of the universal nucleotidesdiscussed above.

The polymerase may replace the one or more nucleotide species with adifferent nucleotide species which comprises a chemical group or atomabsent from the one or more nucleotide species. The chemical group maybe a propynyl group, a thio group, an oxo group, a methyl group, ahydroxymethyl group, a formyl group, a carboxy group, a carbonyl group,a benzyl group, a propargyl group or a propargylamine group.

The polymerase may replace the one or more nucleotide species with adifferent nucleotide species which lacks a chemical group or atompresent in the one or more nucleotide species. The polymerase mayreplace the one or more of the nucleotide species with a differentnucleotide species having an altered electronegativity. The differentnucleotide species having an altered electronegativity preferablycomprises a halogen atom.

The method preferably further comprises selectively removing thenucleobases from the one or more different nucleotides species in themodified polynucleotide.

Other Characterisation Method

In another embodiment, a polynucleotide is characterised by detectinglabelled species that are released as a polymerase incorporatesnucleotides into the polynucleotide. The polymerase uses thepolynucleotide as a template. Each labelled species is specific for eachnucleotide. The polynucleotide is contacted with a pore of theinvention, a polymerase and labelled nucleotides such that phosphatelabelled species are sequentially released when nucleotides are added tothe polynucleotide(s) by the polymerase, wherein the phosphate speciescontain a label specific for each nucleotide. The polymerase may be anyof those discussed above. The phosphate labelled species are detectedusing the pore and thereby characterising the polynucleotide. This typeof method is disclosed in European Application No. 13187149.3 (publishedas EP 2682460). Any of the embodiments discussed above equally apply tothis method.

Kits

The present invention also provides a kit for characterising a targetpolynucleotide. The kit comprises a pore of the invention and thecomponents of a membrane. The membrane is preferably formed from thecomponents. The pore is preferably present in the membrane. The kit maycomprise components of any of the membranes disclosed above, such as anamphiphilic layer or a triblock copolymer membrane.

The kit may further comprise a polynucleotide binding protein.

The kit may further comprise one or more anchors for coupling thepolynucleotide to the membrane.

The kit is preferably for characterising a double strandedpolynucleotide and preferably comprises a Y adaptor and a hairpin loopadaptor. The Y adaptor preferably has one or more helicases attached andthe hairpin loop adaptor preferably has one or more molecular brakesattached. The Y adaptor preferably comprises one or more first anchorsfor coupling the polynucleotide to the membrane, the hairpin loopadaptor preferably comprises one or more second anchors for coupling thepolynucleotide to the membrane and the strength of coupling of thehairpin loop adaptor to the membrane is preferably greater than thestrength of coupling of the Y adaptor to the membrane.

The kit of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides orvoltage or patch clamp apparatus. Reagents may be present in the kit ina dry state such that a fluid sample resuspends the reagents. The kitmay also, optionally, comprise instructions to enable the kit to be usedin the method of the invention or details regarding for which organismthe method may be used.

Apparatus

The invention also provides an apparatus for characterising a targetpolynucleotide. The apparatus comprises a plurality of pores of theinvention and a plurality of membranes. The plurality of pores arepreferably present in the plurality of membranes. The number of poresand membranes is preferably equal. Preferably, a single pore is presentin each membrane.

The apparatus preferably further comprises instructions for carrying outthe method of the invention. The apparatus may be any conventionalapparatus for polynucleotide analysis, such as an array or a chip. Anyof the embodiments discussed above with reference to the methods of theinvention are equally applicable to the apparatus of the invention. Theapparatus may further comprise any of the features present in the kit ofthe invention.

The apparatus is preferably set up to carry out the method of theinvention.

The apparatus preferably comprises:

a sensor device that is capable of supporting the plurality of pores andmembranes and being operable to perform polynucleotide characterisationusing the pores and membranes; and

at least one port for delivery of the material for performing thecharacterisation.

Alternatively, the apparatus preferably comprises:

a sensor device that is capable of supporting the plurality of pores andmembranes being operable to perform polynucleotide characterisationusing the pores and membranes; and

at least one reservoir for holding material for performing thecharacterisation.

The apparatus more preferably comprises:

a sensor device that is capable of supporting the membrane and pluralityof pores and membranes and being operable to perform polynucleotidecharacterising using the pores and membranes;

at least one reservoir for holding material for performing thecharacterising;

a fluidics system configured to controllably supply material from the atleast one reservoir to the sensor device; and

one or more containers for receiving respective samples, the fluidicssystem being configured to supply the samples selectively from one ormore containers to the sensor device.

The apparatus may be any of those described in International ApplicationNo. PCT/GB08/004127 (published as WO 2009/077734), PCT/GB10/000789(published as WO 2010/122293), International Application No.PCT/GB10/002206 (published as WO 2011/067559) or InternationalApplication No. PCT/US99/25679 (published as WO 00/28312).

The following Example illustrates the invention.

Example 1

This Example describes the use of a helicase—T4Dda—E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutationsE94C/C109A/C136A/A360C) to control the movement of DNA construct X(shown in FIG. 1) through a number of different MspA nanopores. All ofthe nanopores tested exhibited changes in current as the DNAtranslocated through the nanopore.

Materials and Methods

Prior to setting up the experiment, DNA construct X or Y (see FIGS. 1and 2 respectively for diagram and sequences used in constructs X and Y,final concentration added to the nanopore system 0.1 nM) waspre-incubated at room temperature for five minutes with T4Dda—E94C/C109A/C136A/A360C (final concentration added to the nanoporesystem 10 nM, SEQ ID NO: 24 with mutations E94C/A360C which was providedin buffer (253 mM KCl, 50 mM potassium phosphate, pH 8.0, 2 mM EDTA)).After five minutes, TMAD (100 μM final concentration added to thenanopore system) was added to the pre-mix and the mixture incubated fora further 5 minutes. Finally, MgCl2 (1 or 2 mM final concentration addedto the nanopore system), ATP (2 mM final concentration added to thenanopore system) and KCl (500 mM final concentration added to thenanopore system) were added to the pre-mix.

Electrical measurements were acquired from single MspA nanopores (pleasesee table 5 below for those tested) inserted in block co-polymer inbuffer (500 mM KCl, 25 mM potassium phosphate pH 8.0). After achieving asingle pore inserted in the block co-polymer, then buffer (1 mL, 500 mMKCl, 25 mM potassium phosphate) was flowed through the system to removeany excess MspA nanopores. The enzyme (T4 Dda—E94C/C109A/C136A/A360C, 10nM final concentration), DNA construct X or Y (0.1 nM finalconcentration), fuel (MgCl2 1 or 2 mM final concentration, ATP 2 mMfinal concentration) pre-mix (150 μL total) was then flowed into thesingle nanopore experimental system and the experiment run with thefollowing protocol −120 mV for 900 s, −180 mV for 2 seconds, 0 mV for 2s and then return to 120 mV for 900 s (this flip protocol was repeated 8times) and helicase-controlled DNA movement was monitored.

Results

The MspA nanopores which were tested are shown in the table 3 below.Helicase controlled DNA movement was observed for DNA constructs X and Y(FIGS. 1 and 2 respectively) using T4 Dda—E94C/C109A/C136A/A360C (seetable 5 for which figures correspond to which nanopore mutants tested).

TABLE 3 Mutant MspA Nanopore Final Mg²⁺ (SEQ ID NO: 2 with the specifiedDNA concentration Figure Entry mutations) Construct (mM) Number 1 MspA -(G75S/G77S/L88N/D90N/ X 1 3 D91N/D118R/Q126R/D134R/E139K)8 2 MspA -(G75S/G77S/L88N/D90N/ Y 2 4 D91N/A96D/D118R/Q126R/D134R/E139K)8 3 MspA -(G75S/G77S/L88N/D90N/ X 1 5 D91N/N102G/D118R/Q126R/D134R/E139K)8 4MspA - (G75S/G77S/L88N/D90N/ X 1 6 D91N/S103A/D118R/Q126R/D134R/E139K)85 MspA - (G75S/G77S/L88N/D90N/ X 1 7D91N/N108S/D118R/Q126R/D134R/E139K)8 6 MspA - (G75S/G77S/L88N/D90N/ X 18 D91N/N108P/D118R/Q126R/D134R/E139K)8 7 MspA -(G75S/G77S/L88N/D90N/D9IN/ X 1 9 A96D/N108P/D118R/Q126R/D134R/E139K)8 8MspA - (G75S/G77S/L88N/D90N/D9IN/ X 1 10A96D/N108A/D118R/Q126R/D134R/E139K)8 9 MspA - (G75S/G77S/L88N/I89F/ X 111 D90N/D91N/D118R/Q126R/D134R/E139K)8 10 MspA - (G75S/G77S/D90N/D91N/ X1 12 D118R/Q126R/D134R/E139K)8 11 MspA - (G75S/G77S/L88K/D90N/ X 1 13D91N/I105E/D118R/Q126R/D134R/E139K)8 12 MspA - (G75S/G77S/L88N/D90N/ X 114 D91N/D118G/Q126R/D134R/E139K)8 13 MspA - (G75S/G77S/L88N/D90N/ X 1 15D91N/D118N/Q126R/D134R/E139K)8 14 MspA - (G75S/G77S/L88N/D90N/ X 1 16D91N/D118R/D134R/E139K)8 19 MspA - (G75S/G77S/L88K/D90N/D9IN/ X 1 17N108E/D118R/Q126R/D134R/E139K)8 21 MspA - (G75S/G77S/L88N/D90N/D9IN/ Y 218 T95E/P98K/D118R/Q126R/D134R/E139K)8 22 MspA - (G75S/G77S/L88N/D90N/ X1 19 D91N/D93N/D118R/Q126R/D134R/E139K)8

Entry 22 (FIG. 19) shows the MspA mutant MspA—(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8 which has substitutedthe aspartic acid at position 93 with a glycine. Helicase-controlled DNAmovement was observed when DNA translocated through this mutant.However, entry 1 (FIG. 3) has position 93 changed back to an asparticacid MspA—(G75S/G77 S/L88N/D90N/D91N/D118R/Q126R/D134R/E139K)8 (which isthe same amino acid that is present in the wild-type MspA) and this alsoexhibited helicase-controlled DNA movement. Therefore, it was possibleto put the aspartic acid back into the nanopore amino acid sequence andhelicase-controlled DNA movement was still observed.

1. A mutant Msp monomer comprising a variant of the sequence shown inSEQ ID NO: 2, wherein the variant: (a) does not comprise aspartic acid(D) at position 90; (b) does not comprise aspartic acid (D) at position91; (c) comprises aspartic acid (D) or glutamic acid (E) at position 93;and (d) comprises one or more modifications which decrease the netnegative charge of the inward facing amino acids in the cap formingregion and/or the barrel forming region of the monomer.
 2. A mutantmonomer according to claim 1, wherein the cap forming region comprisesamino acids 1 to 72 and 122 to 184 of SEQ ID NO:
 2. 3. A mutant monomeraccording to claim 1, wherein the barrel forming region comprises aminoacids 73 to 82 and 112 to 121 of SEQ ID NO:
 2. 4. A mutant monomeraccording to claim 1, wherein the inward facing amino acids in the capforming region are V9, Q12, D13, R14, T15, W40, I49, P53, G54, D56, E57,E59, T61, E63, Y66, Q67, I68, F70, P123, I125, Q126, E127, V128, A129,T130, F131, S132, V133, D134, S136, G137, E139, V144, H148, T150, V151,T152, F163, R165, I167, S169, T170 and S173.
 5. A mutant monomeraccording to claim 1, wherein the inward facing amino acids in thebarrel forming region are S73, G75, G77, N79, S81, G112, S114, S116,D118 and G120.
 6. A mutant monomer according to claim 1, wherein the oneor more modifications are one or more deletions of negatively chargedamino acids or one or more substitutions of negatively charged aminoacids with one or more positively charged, uncharged, non-polar and/oraromatic amino acids.
 7. A mutant monomer according to claim 6, whereinthe one or more negatively charged amino acids are substituted withalanine (A), valine (V), asparagine (N) or glycine (G).
 8. A mutantmonomer according to claim 1, wherein the one or more modifications areone or more introductions of positively charged amino acids.
 9. A mutantmonomer according to claim 6, wherein the one or more positively chargedamino acids are histidine (H), lysine (K) and/or arginine (R).
 10. Amutant monomer according to claim 1, wherein the one or moremodifications are one or more chemical modifications of one or morenegatively charged amino acids which neutralise their negative charge.11. A mutant monomer according to claim 1, wherein the one or moremodifications reduce the net negative charge at one or more of positions118, 126, 134 and
 139. 12. A mutant monomer according to claim 1,wherein (i) the variant comprises a positively charged amino acid at oneor more of positions 114, 116, 120, 123, 70, 73, 75, 77 and 79, (ii) thevariant comprises a positively charged amino acid at one or more ofpositions 123, 125, 127 and 128; (iii) the variant comprises apositively charged amino acid at one or more of positions 129, 132, 136,137, 59, 61 and 63; (iv) the variant comprises a positively chargedamino acid at one or more of positions 137, 138, 141, 143, 45, 47, 49and 51; (v) the variant does not comprise aspartic acid (D) or glutamicacid (E) at one or more of positions 118, 126, 134 and 139; (vi) thevariant comprises arginine (R), glycine (G) or asparagine (N) at one ormore of positions 118, 126, 134 and 139; (vii) the variant comprisesD118R, Q126R, D134R and E139K; (viii) the variant comprises serine (S),glutamine (Q), leucine (L), methionine (M), isoleucine (I), alanine (A),valine (V), glycine (G), phenylalanine (F), tryptophan (W), tyrosine(Y), histidine (H), threonine (T), arginine (R), lysine (K), asparagine(N) or cysteine (C) at position 90 and/or position 91; (ix) the variantcomprises asparagine (N) at position 90 and/or position 91; (x) thevariant comprises one or more of: (e) serine (S) at position 75; (f)serine (S) at position 77; and (g) asparagine (N) or lysine (K) atposition 88; (xi) the variant comprises G75S, G77S and L88K or G75S,G77S and L88N; (xii) the variant comprises G75S, G77S, L88N, D90N, D91N,D118R, Q126R, D134R and E139K; and/or (xiii) the variant furthercomprises one or more of: (h) phenylalanine (F) at position 89; (i)glutamic acid (E) at position 95 and lysine (K) at position 98; (j)aspartic acid (D) at position 96; (k) glycine (G) at position 102; (l)alanine (A) at position 103; and (m) alanine (A), serine (S) or proline(P) at position
 108. 13-32. (canceled)
 33. A pore derived from Mspcomprising at least one mutant monomer according to claim
 1. 34. A poreaccording to claim 33, wherein the pore comprises eight mutant monomersaccording to claim 1 and wherein at least one of them differs from theothers.
 35. A method of characterising a target polynucleotide,comprising: a) contacting the polynucleotide with a pore according toclaim 31 such that the polynucleotide moves through the pore; and b)taking one or more measurements as the polynucleotide moves with respectto the pore, wherein the measurements are indicative of one or morecharacteristics of the polynucleotide, and thereby characterising thetarget polynucleotide.
 36. A method according to claim 35, wherein theone or more characteristics are selected from (i) the length of thepolynucleotide, (ii) the identity of the polynucleotide, (iii) thesequence of the polynucleotide, (iv) the secondary structure of thepolynucleotide and (v) whether or not the polynucleotide is modified.37. A method according to claim 35, wherein the one or morecharacteristics of the polynucleotide are measured by electricalmeasurement and/or optical measurement.
 38. (canceled)
 39. A methodaccording to claim 35, wherein step a) further comprises contacting thepolynucleotide with a polynucleotide binding protein such that theprotein controls the movement of the polynucleotide through the pore.40. (canceled)
 41. A method according to claim 39, wherein thepolynucleotide binding protein is a helicase or is derived from ahelicase. 42-51. (canceled)
 52. A pore according to claim 33, whereinthe pore comprises eight identical mutant monomers according to claim 1.